Substrates, devices, and methods for cellular assays

ABSTRACT

The present invention relates to the field of molecular diagnostics. In particular, the present invention provided improved substrates and methods of using liquid crystals and other biophotonically based assays for quantitating the amount of an analyte in a sample. The present invention also provides materials and methods for detecting non-specific binding of an analyte to a substrate by using a liquid crystal or other biophotonically based assay formats.

This application claims the benefit of U.S. Prov. Appl. 60/836,109,filed Aug. 7, 2006, the contents of which are incorporated by referencein their entirety.

This application was made with the support of Nat'l Institute of GeneralMedical Sciences (NIGMS) grant 2R44GM069026-03. The government may havecertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the fields of molecular biology,cellular biology, developmental biology, stem cell differentiation,immunology, oncology, general laboratory sciences and microbiology, andin particular to methods and compositions based on liquid crystal assaysand other biophotonically based assays for detecting and quantifying thenumber of cells present on a test surface or within a test substrate andthe proliferation, death or movement of cells under control conditionsand in response to chemotactic and other cytoactive (including compoundsthat are chemokinetic but not chemotactic and agents that inhibit cellmigration) agents. Additionally, the present invention describes a novelbiophotonic approach for the detection and quantification of enzymaticactivity.

BACKGROUND OF THE INVENTION

Every year cancer claims the lives of hundreds of thousands of peopleworldwide. The populations of many of the heavily industrializedcountries are particularly susceptible to cancer induced morbidity andmortality. In fact, cancer is the second leading cause of death inindustrialized nations. For example, prostate cancer is the second mostcommon malignancy in men. It is estimated that in 2002 in the UnitedStates nearly 180,000 men will be diagnosed with prostate cancer. Breastcancer is the most common female malignancy in most industrializedcountries, and in the United States it is estimated that breast cancerwill affect about 10% of women during their lives. Approximately 30 to40% of women with operable breast cancer eventually develop metastasesdistant from the primary tumor.

Metastasis, the formation of secondary tumors in organs and tissuesremote from the site of the primary tumor, is the main cause oftreatment failure and death for cancer patients. Indeed, thedistinguishing feature of malignant cells is their capacity to invadesurrounding normal tissues and metastasize through the blood andlymphatic systems to distant organs. Cancer metastasis is a complexprocess by which certain cancer cells acquire substantial geneticmutations and perturbed signal cascades that allow them to leave theprimary tumor mass and establish secondary tumors at distant sites.Metastatic cancer cells break adhesions with neighboring cells, dissolvethe extracellular matrix, migrate and invade surrounding tissue, travelvia the circulatory system, invade, survive and proliferate in newsites. Unfortunately, the molecular mechanisms that promote and restrainthe metastatic spread of cancer cells have yet to be clearly identified.

Medical researchers have made considerable efforts to understand whetherchemotactic agents are involved in metastasis and why particular cancerspreferentially metastasize to certain sites. Breast cancer, for example,favors metastasizing to regional lymph nodes, bone marrow, and lung andliver tissues. Prostate cancer-favors metastasizing to bone marrow.Several theories have been advanced to explain the preferentialmetastasis of certain cancers.

It has recently been shown that one important property of highlymetastatic cells is their ability to respond to chemotactic agents suchas paracrine and autocrine motility factors. For example, recent workdone by Muller et al. provides evidence for chemotactic homing of breastcancer to metastatic sites. (Muller et al. “Involvement of chemokinereceptors in breast cancer metastasis,” Nature, 410:50-56 [2001]); Seealso, M. More, “The role of chemoattraction in cancer metastases,”Bioessays, 23:674-676 [2001]). Muller et al. findings indicate thatCXCR4 and CCR7 chemokine receptors are found on breast cancer cells andthat ligands for these receptors are highly expressed at sitesassociated with preferential breast cancer metastases.

Many conventional assay methods have been adapted for studying theeffects of chemotactic agents on cancer and other cells of interest(e.g., densitometric, analyses of membrane filters, visible spectrum orspectrophotometric ELISA microplate readers, fluorescence microplatereaders, scintillation counters, and photoluminescence readers). Each ofthese methods has particular advantages and disadvantages. Onedisadvantage found in each of these methods is the requirement that thecells of interest be “tagged” with dyes, fluorescing agents, orradioisotopes, in order to observe the cellular responses to chemicalagents. Extrinsic cell labeling techniques add to the expense andcomplexity of the existing assay methods and often require the expertiseof highly skilled technicians.

An important property of metastatic cells is their ability to produceproteases, such as Matrix Metalloproteinases (MMPs) that are capable ofdigesting constituents of the extracellular matrix. The elaboration ofthese proteases facilitates their invasion of tissues. The role ofproteases in the metastatic process using in vitro and in vivo systemsas well as their quantification for use as a prognostic indicator formetastatic potential has been widely reported. The amount of a givenprotease present can be measured using ELISA but this requires aspecific antibody capable of reacting with the protease from a givenspecies. Another drawback of ELISA is that it measures the total amountof a given protease and does not discriminate between proenzyme,activated enzyme or inhibitor complexed enzyme. For example, theactivation state of MMP's in the cellular environment is tightlyregulated by Tissue Inhibitors of Metalloproteinases (TIMPs). Zymography(to measure proteases) and reverse zymography (to measure TIMPs), arewidely used methods that involve gel electrophoresis combined withenzymatic digestion of an appropriate substrate. Both the proenzyme andactive forms of proteases can be distinguished on the basis of molecularweight. Unfortunately, standard zymographic methods are laboriousrequiring many preparative steps (Hawkes S P, Li H, Taniguchi T.)Zymography and reverse zymography for detecting MMPs and TIMPs. InMatrix Metalloproteinase Protocols. Volume 151 of Methods in MolecularBiology. Ian Clark ed. Humana Press. Totowa N.J. 2001. pp 399-410).

Other assays used include a variety of protease assays includingquantifying radiolabelled collagen fragments released by enzymaticcleavage of a radiolabelled substrate, and the measurement offluorescence produced when a fluorescently autoquenched fluorescentsubstrate undergoes digestion and creates an increase in quantifiablefluorescent signal. These methods do not allow discrimination betweenproteases however (Cawston T E, Koshy P, Rowan A D. Assay of matrixmetalloproteinases against matrix substrates. In MatrixMetalloproteinase Protocols. Volume 151 of Methods in Molecular Biology.Ian Clark ed. Humana Press. Totowa N.J. 2001. pp 389-397).

What are needed are assay devices and systems for detecting andquantifying cell number and identifying their spatial location. Otheruseful features include the ability to identify and quantify proteasesand protease inhibitors, without the use of extrinsic cell labelingtechniques, in a robust and easier to use method which allows forenhanced evaluation of samples.

SUMMARY OF THE INVENTION

The present invention relates to the fields of molecular biology,cellular biology, immunology, oncology, developmental biology, stem celldifferentiation, general laboratory sciences and microbiology, and inparticular to methods and compositions based on liquid crystal assaysand other biophotonically based assays for detecting and quantifying thenumber of cells present on a substrate (allows for the quantitation ofcell adhesion and cell proliferation) as well as direct quantificationof proliferation, cell death, differentiation, or cell migration on asurface or through an extracellular matrix (cell invasion) under controlconditions and in response to the presence of chemotactic, growth,differentiation enhancing and other cytoactive (accounts forchemokinetic agents and agents that inhibit cell migration) agents.

Accordingly, in some embodiments, the present invention provides anassay apparatus comprising a surface having at least one discreet assayregion, the discrete assay region comprising at least one cell seedingregion and at least one test compound formulated for controlled release.In some embodiments, the test compound formulated for controlled releaseis provided in a matrix. In some preferred embodiments, the matrix is apolymer. The present invention is not limited to the use of anyparticular type of polymer. Indeed, the use of a variety of polymers iscontemplated, including, but not limited to, chitosan,chitosan-alginate, poly(N-isopropylacrylamide) hydrogels, lipidmicrospheres, copolymers of polylactic and polyglycolic acid, dextranhydrogels, and poly(ethylene glycol) hydrogels. In some embodiments, thematrix further comprises an extracellular matrix component. The presentinvention is not limited to any particular extracellular matrixcomponents. Indeed, the use of a variety of extracellular matrixcomponents is contemplated, including, but not limited to, collagen,vitronectin, fibronectin, and laminin. In some preferred embodiments,the cell seeding region comprises an extracellular matrix component. Thepresent invention is not limited to the use of any particular testcompound. Indeed, the present invention contemplates the use of avariety of test compounds, including, but not limited to, polypeptides,sugars, amino acids and small molecule organic compounds. The presentinvention is not limited to the use of any particular polypeptides.Indeed, the use of a variety polypeptides is contemplated, including,but not limited to integrin binding sequences and growth factors. Thepresent invention is not limited to the use of any particularcarbohydrates. Indeed, the use of a variety of carbohydrates iscontemplated, including, but not limited to, glucose, fructose, sucrose,galactose and derivatives thereof. The present invention is not limitedto the use of any particular small molecule organic compounds. Indeed, avariety find use in the present invention, including, but not limitedto, steroids, immunomodulators, hormones, antineoplastic drugs,antimetabolites, chemotherapeutic agents, antimicrobial drugs, NTHEs,vasodialators, beta-adrenergic blockers, diuretics, anesthetics,antidepressants, sedatives, tranquilizers, vasoconstrictors, anti-ulcerdrugs, stimulants, antihypertensive drugs and cholesterol loweringdrugs. In some preferred embodiments, the test compound is suspected ofpromoting or inhibiting the movement of cells. The assay regions of thedevices of the present invention may be configured for a variety ofreadouts, including, but not limited to, calorimetric, fluorimetric,optical density, liquid crystal, and light scattering readouts. In someembodiments, the at least one cell seeding region contains at least onecell. In some embodiments, the at least one assay region is configuredto orient mesogens. In some embodiments the devices comprise at leastone reservoir. In some embodiments, the at least one reservoir isfluidically connected to at least one microfluidic channel. In somepreferred embodiments, the at least one reservoir fluidically contactsthe at least one assay region. In some embodiments, the apparatuscomprises about 6, 12, 24, 36, 96, 384, or 1536 assay regions. In someembodiments, the about 6, 12, 24, 36, 96, 384, or 1536 regions arearranged in an array of a plurality of rows and columns. In somepreferred embodiments, the array of assay regions is configured tocorrespond to the reading positions of a plate reader device. In someembodiments, the apparatus comprises two or more test compoundsformulated for controlled release.

In some embodiments, the apparatus further comprising at least one wellhaving bottom and side surfaces, wherein the at least one discrete assayregion is located on the bottom surface of the well, and wherein thematrix is located in the well. The present invention is not limited toany particular matrix location. In some embodiments, the matrix islocated on the side surface of the well. In other embodiments, thematrix is located on the bottom of the well. In other embodiments, thematrix is located in a discrete region of the well. In some embodiments,the discrete region is on the bottom of the well. In other embodiments,the discrete region is on the side of the well.

In some embodiments, the present invention provides methods comprisinga) providing cells and an assay apparatus comprising a surface having atleast one discrete assay region, the discrete assay region comprising atleast one cell seeding region and at least one test compound formulatedfor controlled release; b) contacting the cell seeding region with thecells; c) culturing the cells under conditions that the test compound isreleased; and d) assaying the response of the cells to the testcompound. In some embodiments, the surface comprises a plurality ofdiscrete assay regions arranged in an array. In some embodiments, thesurface comprises about 6, 12, 24, 36, 96, 384, or 1536 assay regions.In some embodiments, the about 6, 12, 24, 36, 96, 384, or 1536 assayregions are arranged in an array of a plurality of rows and columns. Insome preferred embodiments, the array of the assay regions is configuredto correspond to the reading positions of a plate reader device. Asdescribed above, in some embodiments, the test compound formulated forcontrolled release is provided in a matrix. In some embodiments, thetest compound is selected from the group consisting of polypeptides,sugars, amino acids and small molecule organic compounds. In someembodiments, the test compound is suspected of promoting or inhibitingmovement of the at least one cell. In some embodiments, the assayregions are configured for readouts selected from the group consistingof calorimetric, fluorimetric, optical density, and light scatteringreadouts. In some embodiments, the at least one cell seeding regioncontains at least one cell. In some embodiments, the at least one assayregion is configured to orient mesogens. In some embodiments, thesurface comprises reservoirs and microfluidic channels. Also, asdescribed above, the matrix can be provided in a variety of location.

The present invention also provides kits comprising: a) an assayapparatus comprising a surface having at least one discrete assayregion, the discrete assay region comprising at least one cell seedingregion; b) unpolymerized matrix material; and c) instructions forpolymerizing the matrix material in the presence of the at least onetest compound, applying the matrix material to the assay apparatus, andculturing cells in the assay apparatus. The assay devices in the kitsare substantially as described above. As above, the kits find use forthe detection and analysis of variety of cells and test compounds.

In some embodiments, the present invention provides devices forfacilitating the seeding of cells in a multiwell plate comprising aplurality of cylinders sized to be inserted into individual wells of amultiwell plate, the cylinders movably connected to at least onehorizontal member so that the cylinders can be positioned in individualwells in the multiwell plate. In some embodiments, the movableconnection allows for horizontal movement of the cylinders. In otherembodiments, the moveable connection allows for vertical movement of thecylinders. In some embodiments, the cylinders are sized to be insertedinto a well of a multiwell plate selected from the group consisting of6, 12, 24, 36, 96, 384, or 1536 well multiwell plates.

In other embodiments, the present invention provides devices forfacilitating the seeding of cells in a multiwell plate comprising aninsert sized to be inserted into individual wells of multiwell plate,the insert comprising a substantially circular surface having therein anopening so that when the insert is positioned in the well the bottomsurface of the well is exposed by the opening in the insert, the insertfurther comprising a lift piece so that the insert can be lifted fromthe well. In some preferred embodiments, the inserts are sized to beinserted into a well of a multiwell plate selected from the groupconsisting of 6, 12, 24, 36, 96, 384, or 1536 well multiwell plates.

In other embodiments, the present invention provides methods of assayingcell migration comprising: a) providing cells and an assay device; b)seeding cells in a discrete area of the assay device; c) assaying cellmovement with a plate reading device. In some embodiments, the assaydevice is a multiwell plate. In some embodiments, the assay device is aslide comprising multiple discrete assay regions. In some preferredembodiments, the plate reading device assays the presence of cellswithin discrete regions of the assay device. In some embodiments, thediscrete regions are concentric circles. In some embodiments, themultiwell plate comprises asymmetrically masked wells. In otherembodiments, the plate reader asymmetrically samples individual wells inthe multiwell plate. In some embodiments, the multiple discrete assayregions are asymmetrically masked. In other embodiments, the platereader asymmetrically samples individual assay regions on the slide.

In still other embodiments, the present invention provides methods ofanalyzing surfaces comprising: a) providing a plate reading device andan article having a coated surface; b) measuring optical density atmultiple discrete regions on the coated surface; and c) comparing theoptical density at the multiple discrete regions to determine theuniformity of the coated surface. In some embodiments, the methodsfurther comprise discarding articles that have less than a predeterminedthreshold of uniformity. In some embodiments, the plate reading deviceis configured to provide readings from about 6 to about 2000 discreteregions. In other embodiments, the methods further comprise presentingthe comparisons graphically.

In other embodiments, the present invention provides methods foranalyzing a lipid membrane containing entity comprising: a) providing:i) a sample suspected of containing of a biological entity with a lipidmembrane; ii) a detection device comprising a substrate comprising atleast one detection region; iii) mesogens; b) contacting the detectionregion with the sample; c) contacting the substrate with the mesogens,wherein the presence of the biological entity with a lipid membrane isindicated by a change in the mesogens over the detection regions andwherein the change is independent of the presence of an additionalhomeotropic director on the detection region. The present invention isnot limited to any particular in the mesogens. In some embodiments, thechange in the mesogens is selected from the group consisting of a changein color, a change in texture, a change in tilt, and homeotropicorientation. The present invention is not limited to the analysis of anyparticular biological entity having a lipid membrane. Indeed, theanalysis of a variety of such entities is contemplated, including, butnot limited to cells, a bacteria, Mycoplasma, viruses, and liposomes orcombinations thereof. The present invention is not limited to the use ofany particular substrate. Indeed, the use of a variety of substrates iscontemplated, including, but not limited to metal films, glass, silicon,diamond and polymeric materials. The use of a variety of polymericmaterials is contemplated, including, but not limited to, polyurethane,PDMS, polyimide, polystyrene, polycarbonate and polyisocyanoacrylate.The present invention is not limited to the use of any particularmesogens. Indeed, the sue of a variety of mesogens is contemplated,including, but not limited to, 4-cyano-4′-pentylbiphenyl,N-(4-methoxybenzylidene)-4-butlyaniline and combinations thereof. Insome embodiments, the detection region further comprises a recognitionmoiety that recognizes the biological entity. The present invention isnot limited to any particular recognition moiety. Indeed, the use of avariety of recognition components is contemplated, including, but notlimited to antigen binding proteins and nucleic acids. In some preferredembodiments, the antigen binding protein is an immunoglobulin. In someembodiments, the substrate comprises a plurality of detection regions.In some embodiments, the plurality of detection regions has the samerecognition moiety bound thereto. In other embodiments, the plurality ofdetection regions has different recognition moieties bound thereto. Infurther embodiments, the detection device further comprises a secondsubstrate arranged opposite the first substrate to form a cell. In someembodiments, the change in the mesogens is detected by viewing thedetection device between cross polar lenses. In some preferredembodiments, the detection region does not homeotropically orientmesogens in the absence of virus. The present invention is not limitedto the analysis of any particular type of sample. Indeed, the use of avariety of samples is contemplated, including biological fluids, tissuehomogenates, feces, vesicular fluids, swabs of orifices or tissues, andmedia in which virus has been cultured or prepared. In some preferredembodiments, the biological fluid is selected from the group consistingof cerebral-spinal fluid, urine, serum, plasma, nasal secretions,sputum, semen and saliva. In some embodiments, the homeotropic orderingis observed within 48 hours of the application of the sample to thedetection region.

In some embodiments, the present invention provides devices for thedetection of an entity comprising a lipid membrane, the devicecomprising a first substrate comprising at least one detection regionhaving at least one recognition moiety specific for the entitycomprising a lipid membrane immobilized thereon, wherein the detectionregion does not homeotropically orient an added mesogen in the absenceof the virus. In some embodiments, the first substrate comprises aplurality of detection regions. In preferred embodiments, the devicescomprise the features described in more detail above.

In some embodiments, the present invention provides kits comprising: a)a device for the detection of a entity comprising a lipid membranecomprising a first substrate comprising at least one detection regionhaving a first recognition moiety specific for the entity comprising alipid membrane immobilized thereon, wherein the detection region doesnot homeotropically orient an added mesogen in the absence of the entitycomprising a lipid membrane; and b) instructions for detection of theentity comprising a lipid membrane. In some embodiments, the kitsfurther comprise a vial containing mesogens. In other embodiments, thekits further comprise a vial comprising the entity comprising a lipidmembrane for use as a positive control. In preferred embodiments, thedevices included in the kit comprise the features described above.

In further embodiments, the present invention provides devices forseeding cells in a well in a multiwell plate comprising, an insert sizedto be inserted into a well of a multiwell plate, the insert having afirst end and a second end and having at least one channel thereinextending from the first end to the second end, the second end having anopening in fluid communication with the channel; wherein when the deviceis inserted into a well of a multiwell plate, the second end contactsthe bottom of the well to seal off a portion of the bottom of the welland the opening provides fluid access to the bottom of the well whereincells can be delivered to the bottom of the well via the at least onechannel. In some embodiments, the insert is cylindrical. In somepreferred embodiments, the second end of the insert comprises aprojection extending from the second end, the projection having aperimeter smaller than the perimeter of the insert. In further preferredembodiments, the projection is circular in shape so that when cells aredelivered to the well, the cells seed in an annular pattern in whichcells are absent from the center of the well. In still other preferredembodiments, the projection is shaped to provide a crescent-shapedopening. In some embodiments the devices further comprise a channelthrough the interior of the insert, wherein the channel provides theopening in the second end. In some preferred embodiments, the opening inthe second end is circular. In further preferred embodiments, the insertis at least partially formed from a material selected from the groupconsisting of PDMS and silicone. In some embodiments, the device issized to be inserted into a well of a multiwell plate selected from thegroup consisting of 6, 12, 24, 96, 394, and 1536 well plates. In furtherembodiments, the device has two channels therein that extend from thefirst end to the second end.

In some embodiments, the present invention provides systems comprising amultiwell plate; at least one insert sized to be inserted into a well ofa multiwell plate, the insert having a first end and a second end andhaving at least one channel therein extending from the first end to thesecond end, the second end having an opening in fluid communication withthe channel; wherein when the insert is inserted into a well of amultiwell plate, the second end contacts the bottom of the well to sealoff a portion of the bottom of the well and the opening provides fluidaccess to the bottom of the well wherein cells can be delivered to thebottom of the well via the at least one channel. In some preferredembodiments, the insert is cylindrical. In further preferredembodiments, the second end of the insert comprises a projectionextending from the second end, the projection having a perimeter smallerthan the perimeter of the insert. In some preferred embodiments, theprojection is circular in shape so that when cells are delivered to thewell, the cells seed in an annular pattern in which cells are absentfrom the center of the well. In other embodiments, the projection isshaped to provide a crescent-shaped opening. In some preferredembodiments, the insert comprises a channel through the interior of theinsert, wherein the channel provides the opening in the second end. Insome embodiments, the opening in the second end is circular. In furtherembodiments, the insert is at least partially formed from a pliablematerial. In other preferred embodiments, the device is sized to beinserted into a well of a multiwell plate selected from the groupconsisting of 6, 12, 24, 96, 394, and 1536 well plates. In still otherembodiments, the systems comprise a plurality of the inserts, whereinthe inserts are provided in a strip and wherein the individual insertsare detachably connected.

In some embodiments, the present invention provides methods of seedingcells in a well of a multiwell plate, comprising a) providing amultiwell plate and an insert sized to be inserted into the multiwellplate, b) inserting the insert into the multiwell plate so that theinsert contacts the bottom of the multiwell plate so that apredetermined portion of the bottom of the well is sealed and a portionof the bottom of the well is exposed to form an exposed portion; and c)applying cells to the well, wherein the cells initially attach to theexposed portion of the well and do not attach to the predeterminedportion of the well. In some embodiments, the methods further comprisethe step of removing the insert. In some embodiments, the methodsfurther comprise the step of assaying the migration of the cells fromthe exposed portion to the predetermined portion. In some preferredembodiments, the predetermined portion is circular in shape so that thecells are seeded in annular pattern. In other preferred embodiments, thepredetermined portion is shaped so that the cells are seeded in acrescent-shaped pattern. In still other preferred embodiments, thepredetermined portion is shaped so that the exposed area is circular andin the middle of the predetermined portion. In some preferredembodiments, the assaying is selected from the group consisting ofcalorimetric, fluorimetric, light scattering, liquid crystal,densitometric, and microscopic assays. In further preferred embodiments,the assays are read by a plate reader. In some embodiments, the methodsfurther comprise the step of contacting the cells with a test compoundthat is suspected of promoting or inhibiting movement of the at leastone cell. In some preferred embodiments, the insert is cylindrical.

In some embodiments, the present invention provides systems for seedingcells on a support comprising: a support; a base having a series of baseopenings therein; and a gasket formed from a pliable material, thegasket having a series of gasket openings therein aligning with theseries of base openings. In some preferred embodiments, the support is aglass slide. In some preferred embodiments, the gasket is formed from amaterial selected from the group consisting of silicone and PDMS. Insome embodiments, the base has two long sides, each of the long sideshaving a channel therein and the system further comprising clips,wherein the clips each have channel extensions, the channel extensionsengageable with the channels so that the support, the gasket, and thebase are securable to create a series of wells on the slidecorresponding to the base and gasket openings.

In some embodiments, the present invention provides systems for seedingcells on a slide comprising: a support; a base having a series ofcircular base openings therein and a channel extending down the longsides of the base; a gasket formed from a pliable material, the gaskethaving a series of gasket openings therein aligning with the series ofbase openings; and two clips comprising channel extensions; wherein thechannel extensions are engageable with the channels in the base so thatthe slide, the gasket, and the base are securable to create a series ofwells on the slide corresponding to the base and gasket openings.

In some embodiments, the present invention provides systems for cellassays comprising: a multiwell plate comprising multiple wells; and amask configured to align with the multiwell plate, wherein the maskmasks a predetermined portion of the wells to provide a masked portionwherein cells are blocked from view and an unmasked portion of the wellswherein cells remain visible. In some embodiments, the mask is polymericand is fixable to the multiwell plate. In other embodiments, the mask isflexible and backed by adhesive so the mask can be secured to themultiwell plate. In some embodiments, the systems further comprise atleast one well insert configured to allow seeding of cells in apredetermined portion of the wells. In some embodiments, the mask masksan area less than the predetermined portion of the wells. In otherembodiments, the mask masks an area equal to the predetermined portionof the wells. In still other embodiments, the mask masks an area greaterthan the predetermined portion of the wells. In some embodiments, thesystems comprise a series of masks having different sized aperturestherein corresponding to the unmasked portions. In some preferredembodiments, the masks are fixable to the multiwell plate.

In some embodiments, the present invention provides methods for assayingcells comprising: seeding cells in a predetermined portion of one ormore wells of a multiwell plate; masking the predetermined portion ofthe one or more wells of a multiwell plate; and assaying movement of thecells out of the predetermined portion of one or more wells of themultiwell plate.

In some embodiments, the present invention provides an insert sized tobe inserted into a well of a multiwell plate comprising first and secondends, wherein the second end is configured to be inserted into the welland contact the bottom of the well to define a cell seeding area.

In some embodiments, the present invention provides a device comprisinga series of inserts configured to be inserted into a well of a multiwellplate, the inserts being detachably connected so that individual insertsmay be removed.

In still other embodiments, the present invention provides an insertremoval tool comprising: an insert end comprising a plurality ofextensions having openings therebetween, the extensions configured tocontact the opposite sides of an insert in a multiwell plate; and ahandle extending from the insert end.

DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic depiction of a nanostamper for use in the presentinvention.

FIG. 1B is a schematic depiction of a negative nanostamper for use inthe present invention.

FIG. 2A is a schematic depiction of random ordering of mesogens.

FIG. 2B is a schematic depiction of ordering of mesogens induced by anelectric field.

FIG. 3 is a schematic depiction of the effect of reducing ordiscontinuing a motive force such as an electric field.

FIG. 4 is a graphical depiction of the relationship between lighttransmission and motive force.

FIG. 5 is a schematic depiction of a device of the present inventionthat incorporates a matrix.

FIG. 6 is a schematic depiction of a device of the present inventionthat incorporates a matrix.

FIG. 7 is a schematic depiction of a device of the present inventionthat incorporates a matrix.

FIG. 8 is a schematic depiction of a device of the present inventionthat incorporates a matrix.

FIG. 9 is a schematic depiction of a device of the present inventionthat incorporates a matrix.

FIG. 10 is a schematic depiction of a device of the present inventionthat incorporates a matrix.

FIG. 11 is a schematic depiction of a device of the present inventionthat incorporates particles on a substrate.

FIG. 12 is a schematic depiction of a device of the present inventionthat incorporates a matrix.

FIG. 13 is a depiction of a cell assay device of the present invention.

FIG. 14 is a schematic depiction of a cell culture device of the presentinvention.

FIG. 15 is a schematic depiction of an assay device of the presentinvention.

FIG. 16 is a schematic depiction of an assay device of the presentinvention.

FIG. 17 is a schematic depiction of an assay device of the presentinvention.

FIG. 18 is a schematic depiction of an assay device of the presentinvention.

FIG. 19 is a schematic depiction of an assay device of the presentinvention.

FIG. 20A is a schematic depiction of an assay device of the presentinvention in use.

FIG. 20B is a schematic depiction of an assay device of the presentinvention in use.

FIG. 20C is a schematic depiction of an assay device of the presentinvention in use.

FIG. 21 is a schematic depiction of an assay device of the presentinvention.

FIG. 22 is a schematic depiction of an assay device of the presentinvention.

FIG. 23 is a schematic depiction of an assay device of the presentinvention.

FIG. 24 is a schematic depiction of an assay device of the presentinvention.

FIG. 25 is a schematic depiction of an assay device of the presentinvention.

FIG. 26 is a schematic depiction of an assay device of the presentinvention.

FIG. 27 is a schematic depiction of an assay device of the presentinvention.

FIG. 28 is a schematic depiction of an assay device of the presentinvention in use.

FIG. 29 is a schematic depiction of an assay device of the presentinvention in use.

FIG. 30 is a schematic depiction of an assay device of the presentinvention.

FIG. 31 is a schematic depiction of an assay device of the presentinvention.

FIG. 32 is a schematic depiction of an assay device of the presentinvention.

FIG. 33 is a schematic depiction of an assay device of the presentinvention.

FIG. 34 is a schematic depiction of an assay device of the presentinvention in use.

FIG. 35 is a schematic depiction of an assay device of the presentinvention.

FIG. 36 is a schematic depiction of an assay device of the presentinvention.

FIG. 37A is a schematic depiction of an assay device of the presentinvention.

FIG. 37B is a schematic depiction of an assay device of the presentinvention.

FIG. 38A is a schematic depiction of an assay device of the presentinvention.

FIG. 38B is a schematic depiction of an assay device of the presentinvention.

FIG. 39 is a schematic depiction of an assay device of the presentinvention.

FIG. 40 is a schematic depiction of an assay device of the presentinvention.

FIG. 41 is a schematic depiction of an assay device of the presentinvention in use.

FIG. 42 is a schematic depiction of an assay device of the presentinvention.

FIG. 43 is a schematic depiction of a plate top of the presentinvention.

FIG. 44 is a schematic depiction of liquid orientation by the plate topof FIG. 43.

FIG. 45 is a schematic depiction of a plate cover for orienting cells inthe center of a well in a multiwell plate.

FIG. 46 is a graph depicting results of quantification of an analyteusing microfluidic channels and liquid crystals.

FIG. 47 is a graph depicting results of an experiment to determine cellnumber with a liquid crystal assay.

FIG. 48 is a schematic depiction of a substrate holder of the presentinvention.

FIG. 49 is graph of the results of a zymography experiment.

FIG. 50 is a schematic depiction of a device for facilitating theseeding of cells in a multiwell plate.

FIG. 51 is a schematic depiction of a device for facilitating theseeding of cells in a multiwell plate.

FIG. 52A is a schematic depiction of an assay device in slide format.

FIG. 52B is a schematic depiction of an assay device in slide format.

FIG. 52C is a schematic depiction of an assay device in slide format.

FIG. 53A is a graph of the optical density of various regions ofgold-coated aluminosilicate glass slide.

FIG. 53B is a graph of the optical density of various regions ofgold-coated soda lime glass slide.

FIG. 54 provides a schematic view of an assay device of the presentinvention demonstrating homeotropic orientation of a liquid crystaldirected by bound virus.

FIG. 55 depicts an insert for seeding cells in a multiwell plate.

FIG. 56 depicts the seeding pattern obtained using the insert depictedin FIG. 55.

FIG. 57 depicts an insert for seeding cells in a multiwell plate.

FIG. 58 depicts the seeding pattern obtained using the insert depictedin FIG. 57.

FIG. 59 depicts an insert for seeding cells in a multiwell plate.

FIG. 60 depicts the seeding pattern obtained using the insert depictedin FIG. 59.

FIG. 61 depicts a mask for use with a multiwell plate

FIG. 62 depicts a device for seeding cells on a substrate.

FIGS. 63 A and B depict a device for removing inserts from a multiwellplate.

FIG. 64 depicts a cell seeding insert of the present invention.

FIG. 65 depicts a mask for a 96-well plate.

FIG. 66 A-D depict features of a mask for a 96-well plate.

DEFINITIONS

As used herein, the term “substrate” refers to material capable ofsupporting associated assay components (e.g., assay regions, mesogensthat constitute the functional units of liquid crystals, cells, testcompounds, etc.). For example, in some embodiments, the substratecomprises a planar (i.e., 2 dimensional) glass, metal, composite,plastic, silica, or other biocompatible or biologically unreactive (orbiologically reactive) composition. In some other embodiments, thesubstrate comprises a porous (e.g., microporous) or structured (i.e., 3dimensional) composition (e.g., sol-gel matrices).

As used herein, the term “mesogen” refers to compounds that form liquidcrystals, and in particular rigid, rodlike or disclike molecules thatare components of liquid crystalline materials.

As used herein, “assay region” refers to a position on a substrateconfigured for the collection of data. In some embodiments, assayregions are configured to order mesogens. In other embodiments, assayregions are configured specifically to not order mesogens. In stillfurther embodiments, assay regions are configured to provide two or moredistinct regions (e.g., optically opaque regions and opticallytransparent regions, regions that are capable of ordering mesogens ofliquid crystal (mesogens) and regions specifically lacking the abilityto order mesogens placed on their surface, and combinations thereof).

As used herein, “array” refers to a substrate with a plurality ofmolecules (e.g., mesogens, recognition moieties) and/or structures(e.g., wells, reservoirs, channels, and the like) associated with itssurface in an orderly arrangement (e.g., a plurality of rows andcolumns). In another sense, the term “array” refers to the orderlyarrangement (e.g., rows and columns) of two or more assay regions on asubstrate.

The term “cell seeding region” as used herein, refers to a portion of anassay region or a substrate that is configured to provide an initialattachment site for one or more cell(s) of interest. In certainpreferred embodiments, the cell seeding region comprises a depression inan assay region of the substrate.

As used herein, “taxis” refers to a response in which the direction ofmovement is affected by an environmental cue. It is clearlydistinguished from a kinesis.

As used herein, “kinesis” refers to alteration in the movement of acell, without any directional bias. Thus speed may increase or decrease(orthokinesis) or there may be an alteration in turning behavior(kiinokinesis).

As used herein, “orthokinesis” refers to kinesis in which the speed orfrequency of movement is increased (positive orthokinesis) or decreased(negative orthokinesis).

As used herein, the term “chemokinesis” refers to a response by a motilecell to a soluble chemical that involves an increase or decrease inspeed (positive or negative orthokinesis) or of frequency of movement ora change in the frequency or magnitude of turning behavior(klinokinesis).

As used herein, the term “chemotaxis” refers to a response of motilecells or organisms in which the direction of movement is affected by thegradient of a diffusible substance. Differs from chemokinesis in thatthe gradient alters probability of motion in one direction only, ratherthan rate or frequency of random motion.

As used herein, the term “neoplasia” refers to abnormal new growth andthus means the same as tumor, which may be benign or malignant. This isnow a general term used interchangeably with the term cancer, for morethan 100 diseases that are characterized by uncontrolled, abnormalgrowth of cells. Neoplastic or cancerous cells can spread locally orthrough the bloodstream and lymphatic systems to other parts of thebody.

As used herein, the term “migration” refers to the passing from onelocation to another. Used to describe the change in position of cells,microorganisms, particles or molecules.

As used herein, “cell movement” refers to any movement or change inshape of a cell including, but not limited to locomotion and cytoplasmicstreaming, etc. As used herein, the term “proliferation” refers to thereproduction or multiplication of similar forms, especially of cells.

As used herein, “contraction” refers to a shortening or reduction insize of a cell. Typically associated with transduction of forces onto orinto a substrate to which the cell is associated.

As used herein, the term “invasion” refers to the movement of cell(s)into a territory of differing composition. In particular it refers tothe use of in vitro assay systems where cells are seeded on onesubstrate and they subsequently move into a 3 dimensional matrix.Ability to “invade” the 3 dimensional matrix is sometimes used as anindicator of malignant potential.

As used herein, the term “phototaxis” refers to movement of a cell ororganism towards (positive phototaxis) or away from a source of light(negative phototaxis).

As used herein, the term “aerotaxis” refers to an organism's movementtoward or away from oxygen as a reaction to its presence. The term ismost often used when discussing aerobes (oxygen-using) versus anaerobes(which don't use oxygen).

As used herein, the term “osmotaxis” refers to movement of a cell ororganism towards (positive osmotaxis) or away from (negative osmotaxis)a source of increased osmotic concentration of solutes.

As used herein, the term “immobilization” refers to the attachment orentrapment, either chemically or otherwise, of a material to anotherentity (e.g., a solid support) in a manner that restricts the movementof the material.

As used herein, the term “surface configured to orient mesogens” refersto surfaces that intrinsically orient mesogens (e.g., throughanisotropic surface features such as obliquely deposited gold or rubbedproteins) and surfaces that are modified to orient liquid crystals byapplication of extrinsic structure or forces, including, but not limitedto particles, electric fields, magnetic fields, or combinations thereof.

As used herein, the term “matrix” refers to any three dimensionalnetwork of materials, including, but not limited to, extracellularmatrices, synthetic or biological polysaccharide matrices, collagenmatrices, matrigel, polymer networks, soft microfabricated structures(e.g., from PDMS), gels of lyotropic liquid crystals, and matricesprepared from bacterial cell secretions. The materials of the matricesmay be chemically crosslinked or physically crosslinked.

As used herein, the terms “material” and “materials” refer to, in theirbroadest sense, any composition of matter.

As used herein the term “polypeptide” is used in its broadest sense torefer to all molecules or molecular assemblies containing two or moreamino acids. Such molecules include, but are not limited to, proteins,peptides, enzymes, antibodies, receptors, lipoproteins, andglycoproteins.

As used herein the term “antigen binding protein” refers to a responseevoked in an animal by an immunogen (antigen), or to proteins selectedin a phage display system, and to proteins derived from such proteins orglycoproteins (e.g., single chain antibodies and F(ab′)2, Fab′ and Fabfragments). An antibody demonstrates binding to the immunogen, or, morespecifically, to one or more epitopes contained in the immunogen. Nativeantibody comprises at least two light polypeptide chains and at leasttwo heavy polypeptide chains. Each of the heavy and light polypeptidechains contains at the amino terminal portion of the polypeptide chain avariable region (i.e., VH and VL respectively), which contains a bindingdomain that interacts with antigen. Each of the heavy and lightpolypeptide chains also comprises a constant region of the polypeptidechains (generally the carboxy terminal portion) which may mediate thebinding of the immunoglobulin to host tissues or factors influencingvarious cells of the immune system, some phagocytic cells and the firstcomponent (C1q) of the classical complement system. The constant regionof the light chains is referred to as the “CL region,” and the constantregion of the heavy chain is referred to as the “CH region.” Theconstant region of the heavy chain comprises a CH1 region, a CH2 region,and a CH3 region. A portion of the heavy chain between the CH1 and CH2regions is referred to as the hinge region (i.e., the “H region”). Theconstant region of the heavy chain of the cell surface form of anantibody further comprises a spacer-transmembranal region (M1) and acytoplasmic region (M2) of the membrane carboxy terminus. The secretedform of an antibody generally lacks the M1 and M2 regions. Therecognition sequence may be derived from the circulating plasma of ananimal or from specific in vitro culture systems (e.g., monoclonalantibodies, recombinant antibodies, and Fabs harvested from eukaryoticand prokaryotic culture systems or phage display systems).

As used herein, the term “non-specific binding” refers to thepositioning (immobilization) of an analyte (target molecule, cell,parasite, virus, bacteria, particle etc) of interest on the surface,wherein the analyte that is not bound by a recognition moiety.

As used herein, the term “analytes” refers to any material that is to beanalyzed. Such materials can include, but are not limited to, ions,molecules, amino acids, polypeptides, nucleic acids, antigens, bacteria,fungi, compounds, viruses, cells, prokaryotic and eukaryotic organisms,multicellular organisms, antibodies, cell parts, and particulate matterin suspension.

As used herein, the term “small organic molecule” refers to any moleculewith low molecular weight (i.e., less than 10,000 atomic mass units andpreferably less than 5,000 atomic mass units). Small organic moleculescan, for example, bind to ligands, interact with ligands, bind toreceptors, bind to nucleic acid, and bind to proteins. Small organicmolecules include, but are not limited to, peptides, polypeptides,steroids, vitamins, and various organic molecules with known biologicalactivity such as those discussed in Section III below and compounds andderivatives related thereto.

As used herein, the term “selective binding” refers to the binding ofone material to another in a manner dependent upon the presence of aparticular molecular structure (i.e., specific binding). For example, areceptor will selectively bind ligands that contain the chemicalstructures complementary to the ligand binding site(s). This is incontrast to “non-selective binding,” whereby interactions are arbitraryand not based on structural compatibilities of the molecules.

As used herein, the term “conformational change” refers to thealteration of the molecular structure of a substance. It is intendedthat the term encompass the alteration of the structure of a singlemolecule or molecular aggregate (e.g., the change in structure of areceptor upon binding a ligand).

As used herein, the term “pathogen” refers to disease causing organisms,microorganisms, or agents including, but not limited to, viruses,bacteria, parasites (including, but not limited to, organisms within thephyla Protozoa, Platyhelminthes, Aschelminithes, Acanthocephala, andArthropoda), fungi, and prions.

As used herein, the term “bacteria” and “bacterium” refer to allprokaryotic organisms, including those within all of the phyla in theKingdom Procaryotae. It is intended that the term encompass allmicroorganisms considered to be bacteria including Mycoplasma,Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms ofbacteria are included within this definition including cocci, bacilli,spirochetes, spheroplasts, protoplasts, etc. “Gram negative” and “Grampositive” refer to staining patterns obtained with the Gram-stainingprocess that is well known in the art (See e.g., Finegold and Martin,Diagnostic Microbiology, 6th Ed. (1982), CV Mosby St. Louis, pp 13-15).

As used herein, the term “polymerization” encompasses any process thatresults in the conversion of small molecular monomers into largermolecules consisting of repeated units. Typically, polymerizationinvolves chemical crosslinking of monomers to one another.

As used herein, the term “membrane receptors” refers to constituents ofmembranes that are capable of interacting with other molecules ormaterials. Such constituents can include, but are not limited to,proteins, lipids, carbohydrates, and combinations thereof.

As used herein, the term “volatile organic compound” or “VOC” refers toorganic compounds that are reactive (i.e., evaporate quickly, explosive,corrosive, etc.), and typically are hazardous to human health or theenvironment above certain concentrations. Examples of VOCs include, butare not limited to, alcohols, benzenes, toluenes, chloroforms, andcyclohexanes.

As used herein, the term “enzyme” refers to molecules or moleculeaggregates that are responsible for catalyzing chemical and biologicalreactions. Such molecules are typically proteins, but can also compriseshort peptides, RNAs, DNAs, ribozymes, antibodies, and other molecules,metals and ions.

As used herein, the term “drug” refers to a substance or substances thatare used to diagnose, treat, or prevent diseases or conditions. Drugsact by altering the physiology of a living organism, tissue, cell, or invitro system that they are exposed to. It is intended that the termencompass antimicrobials, including, but not limited to, antibacterial,antifungal, and antiviral compounds. It is also intended that the termencompass antibiotics, including naturally occurring, synthetic, andcompounds produced by recombinant DNA technology.

As used herein, the term “carbohydrate” refers to a class of moleculesincluding, but not limited to, sugars, starches, cellulose, chitin,glycogen, and similar structures. Carbohydrates can also exist ascomponents of glycolipids and glycoproteins.

As used herein, the term “antigen” refers to any molecule or moleculargroup that is recognized by at least one antibody. By definition, anantigen must contain at least one epitope (i.e., the specificbiochemical unit capable of being recognized by the antibody). The term“immunogen” refers to any molecule, compound, or aggregate that inducesthe production of antibodies. By definition, an immunogen must containat least one epitope (i.e., the specific biochemical unit capable ofcausing an immune response).

As used herein, the term “chelating compound” refers to any compoundcomposed of or containing coordinate links that complete a closed ringstructure. The compounds can combine with metal ions, attached bycoordinate bonds to at least two of the nonmetal ions.

As used herein, the term “recognition moiety” refers to any molecule,molecular group, or molecular complex that is capable of recognizing(i.e., specifically interacting with) a molecule. For example, theligand binding site of a receptor would be considered a molecularrecognition complex. The term “recognition moiety” also encompassesbinding sequences known to bind with a specific target molecule (e.g.,the binding sequence of Raf 1 that specifically binds Ras) and moleculesthat bind metals.

As used herein, the term “cellular binding moiety” refers to anymolecule, molecular group, or molecular complex that binds cells.Examples of cellular binding moieties include, but are not limited to,integrin binding sequences such as RGD sequences and other bindingsequences found in extracellular matrix proteins.

As used herein, the terms “home testing” and “point of care testing”refer to testing that occurs outside of a laboratory environment. Suchtesting can occur indoors or outdoors at, for example, a privateresidence, a place of business, public or private land, in a vehicle, aswell as at the patient's bedside.

As used herein, the term “virus” refers to minute infectious agentswhich, with certain exceptions, are not observable by light microscopy,lack independent metabolism, and are able to replicate only within aliving host cell. The individual particles (i.e., virions) consist ofnucleic acid and a protein shell or coat; some virions also have a lipidcontaining membrane. The term “virus” encompasses all types of viruses,including animal, plant, phage, and other viruses, including thoseincapable of replication without the presence of other viruses.

As used herein, the term “nanostructures” refers to microscopicstructures, typically measured on a nanometer scale. Such structuresinclude various three-dimensional assemblies, including, but not limitedto, liposomes, films, multilayers, braided, lamellar, helical, tubular,pillar like and fiber-like shapes, and combinations thereof. Suchstructures can, in some embodiments, exist as solvated polymers inaggregate forms such as rods and coils. Such structures can also beformed from inorganic materials, such as prepared by the physicaldeposition of a gold film onto the surface of a solid, proteinsimmobilized on surfaces that have been mechanically rubbed, polymericmaterials that have been mechanically rubbed, polymeric or metallicsurfaces into which order has been introduced onto its surface by theuse of micro and nanoabrasive materials (nanoblasting), high pressurewater etching, and polymeric materials that have been molded orimprinted with topography by using a silicon template prepared byelectron beam or other lithographic processes. Extrinsically structuredanisotropic surfaces can also be formed by the placement of submicron to10 μm sized particles (anisometric and/or isometric depending on themethod used) and aligning or partially aligning the particles throughthe use of external fields (including, but not limited to, electricfields, magnetic fields, shear fields and/or fluid flow). It is alsopossible to create an aligned surface using mechanical transfer oforganized or aligned particles (e.g., fabrication with a hydrophobicstamp containing the desired topography). The particles, when depositedonto the surface are organized or aligned such that mesogens containedwithin an overlying liquid crystal are aligned. These particles aredisplaced or reoriented when cells grow on the surface. Alternatively,the stamp can be made from friable materials that are transferred to thesubstrate upon contact with the substrate. Examples of such transferablematerials include, but are not limited to, charcoal, chalk, soapstone,graphite, pumice, other easily fragmented and transferred materials andsynthetic laminated material, prepared such that fracturing layers aredesigned into the material. Nanostructured substrates can also befabricated using scanning probe methods, including atomic forcemicroscopy and scanning tunneling microscopy, as well as x-raylithography, micro/nanoabrasive methods, interferometric opticallithographic methods, and imprinting and embossing (including hot andcold embossing). Similarly, order can be introduced into a particlecovered surface whereby particles are initially randomly positionedacross a surface and an ordered pattern introduced by the selectiveremoval of particles.

As used the term “multilayer” refers to structures comprised of two ormore monolayers. The individual monolayers may chemically interact withone another (e.g., through covalent bonding, ionic interactions, van derWaals' interactions, dipole bonding, hydrogen bonding, hydrophobic orhydrophilic assembly, and steric hindrance) to produce a film with novelproperties (i.e., properties that are different from those of themonolayers alone).

As used herein, the terms “self-assembling monomers” and “lipidmonomers” refer to molecules that spontaneously associate to formmolecular assemblies. In one sense, this can refer to surfactantmolecules that associate to form surfactant molecular assemblies. Theterm “self-assembling monomers” includes single molecules (e.g., asingle lipid molecule) and small molecular assemblies (e.g., polymerizedlipids), whereby the individual small molecular assemblies can befurther aggregated (e.g., assembled and polymerized) into largermolecular assemblies.

As used herein, the term “ligands” refers to any ion, molecule,molecular group, or other substance that binds to another entity to forma larger complex. Examples of ligands include, but are not limited to,peptides, carbohydrates, nucleic acids, antibodies, or any moleculesthat bind to receptors.

As used herein, the terms “organic matrix” and “biological matrix” referto collections of organic molecules that are assembled into a largermulti-molecular structure. Such structures can include, but are notlimited to, films, monolayers, and bilayers. As used herein, the term“organic monolayer” refers to a thin film comprised of a single layer ofcarbon-based molecules. In one embodiment, such monolayers can becomprised of polar molecules whereby the hydrophobic ends all line up atone side of the monolayer. The term “monolayer assemblies” refers tostructures comprised of monolayers. The term “organic polymetric matrix”refers to organic matrices whereby some or all of the molecularconstituents of the matrix are polymerized.

As used herein, the term “linker” or “spacer molecule” refers tomaterial that links one entity to another. In one sense, a molecule ormolecular group can be a linker that is covalently attached to two ormore other molecules (e.g., linking a ligand to a self-assemblingmonomer).

As used herein, the term “bond” refers to the linkage between atoms inmolecules and between ions and molecules in crystals. The term “singlebond” refers to a bond with two electrons occupying the bonding orbital.Single bonds between atoms in molecular notations are represented by asingle line drawn between two atoms (e.g., C—C). The term “double bond”refers to a bond that shares two electron pairs. Double bonds arestronger than single bonds and are more reactive. The term “triple bond”refers to the sharing of three electron pairs. As used herein, the term“ene-yne” refers to alternating double and triple bonds. As used hereinthe terms “amine bond,” “thiol bond,” and “aldehyde bond” refer to anybond formed between an amine group (i.e., a chemical group derived fromammonia by replacement of one or more of its hydrogen atoms byhydrocarbon groups), a thiol group (i.e., sulfur analogs of alcohols),and an aldehyde group (i.e., the chemical group —CHO joined directlyonto another carbon atom), respectively, and another atom or molecule.

As used herein, the term “covalent bond” refers to the linkage of twoatoms by the sharing of two electrons, one contributed by each of theatoms.

As used herein, the term “spectrum” refers to the distribution of lightenergies arranged in order of wavelength.

As used the term “visible spectrum” refers to light radiation thatcontains wavelengths from approximately 360 nm to approximately 800 nm.

As used herein, the term “ultraviolet irradiation” refers to exposure toradiation with wavelengths less than that of visible light (i.e., lessthan approximately 360 nm) but greater than that of X-rays (i.e.,greater than approximately 0.1 nm). Ultraviolet radiation possessesgreater energy than visible light and is therefore, more effective atinducing photochemical reactions.

As used herein, the term “badge” refers to any device that is portableand can be carried or worn by an individual working in an analytedetecting environment.

As used herein, the term “biological organisms” refers to anycarbon-based life forms.

As used herein, the term “in situ” refers to processes, events, objects,or information that are present or take place within the context oftheir natural environment.

As used herein, the term “sample” is used in its broadest sense. In onesense it can refer to a biopolymeric material. In another sense, it ismeant to include a specimen or culture obtained from any source, as wellas biological and environmental samples. Biological samples may beobtained from animals (including humans) and encompass fluids (includinglacrimal and salivary secretions as well as urinary samples), solids,tissues, cells, and gases. Biological samples include blood products,such as plasma, serum and the like. Biological samples also includespecimens obtained in the course of laboratory investigations andinclude cells in media, bacteria, fungi, parasites and/or virus in mediaand particulate matter in suspension. Environmental samples includeenvironmental material such as surface matter, soil, water, crystals andindustrial samples. These examples are not to be construed as limitingthe sample types applicable to the present invention.

As used herein, the term “liquid crystal” refers to a thermodynamicstable phase characterized by anisotropy of properties without theexistence of a three-dimensional crystal lattice, generally lying in thetemperature range between the solid and isotropic liquid phase.

As used herein, “thermotropic liquid crystal” refers to liquid crystalsthat result from the melting of mesogenic solids due to an increase intemperature. Both pure substances and mixtures form thermotropic liquidcrystals.

“Lyotropic,” as used herein, refers to molecules that form phases withorientational and/or positional order in a solvent. Lyotropic liquidcrystals can be formed using amphiphilic molecules (e.g., sodiumlaurate, phosphatidylethanolamine, lecithin). The solvent can be water.

As used herein, the term “heterogeneous surface” refers to a surfacethat orients liquid crystals in at least two separate planes ordirections, such as across a gradient.

As used herein, “nematic” refers to liquid crystals in which the longaxes of the molecules remain substantially parallel, but the positionsof the centers of mass are randomly distributed. Nematic liquid crystalscan be substantially oriented by a nearby surface.

“Chiral nematic,” as used herein refers to liquid crystals in which themesogens are optically active. Instead of the director being heldlocally constant as is the case for nematics, the director rotates in ahelical fashion throughout the sample. Chiral nematic crystals show astrong optical activity that is much higher than can be explained on thebases of the rotatory power of the individual mesogens. When light equalin wavelength to the pitch of the director impinges on the liquidcrystal, the director acts like a diffraction grating, reflecting mostand sometimes all light incident on it. If white light is incident onsuch a material, only one color of light is reflected and it iscircularly polarized. This phenomenon is known as selective reflectionand is responsible for the iridescent colors produced by chiral nematiccrystals.

“Smectic,” as used herein refers to liquid crystals which aredistinguished from “nematics” by the presence of a greater degree ofpositional order in addition to orientational order; the molecules spendmore time in planes and layers than they do between these planes andlayers. “Polar smectic” layers occur when the mesogens have permanentdipole moments. In the smectic A2 phase, for example, successive layersshow anti-ferroelectric order, with the direction of the permanentdipole alternating from layer to layer. If the molecule contains apermanent dipole moment transverse to the long molecular axis, then thechiral smectic phase is ferroelectric. A device utilizing this phase canbe intrinsically bistable.

“Frustrated phases,” as used herein, refers to another class of phasesformed by chiral molecules. These phases are not chiral, however, twistis introduced into the phase by an array of grain boundaries. A cubiclattice of defects (where the director is not defined) exists in acomplicated, orientationally ordered twisted structure. The distancebetween these defects is hundreds of nanometers, so these phases reflectlight just as crystals reflect x-rays.

“Discotic phases” are formed from molecules that are disc shaped ratherthan elongated. Usually these molecules have aromatic cores and sixlateral substituents. If the molecules are chiral or a chiral dopant isadded to a discotic liquid crystal, a chiral nematic discotic phase canform.

As used herein, the term “virus recognition moiety” refers to anycompound that binds specifically to a virus. Examples of “virusrecognition moieties” include, but are not limited to antigen bindingproteins and nucleic acid aptamers.

As used herein, the term “homeotropic director” refers to atopographical feature (e.g., a nanostructure) of a substrate thathomeotropically orients a liquid crystal.

As used herein, the term “pathogen” refers to disease causing organisms,microorganisms, or agents including, but not limited to, viruses,bacteria, parasites (including, but not limited to, organisms within thephyla Protozoa, Platyhelminthes, Aschelminithes, Acanthocephala, andArthropoda), fungi, and prions.

As used herein, the term “bacteria” and “bacterium” refer to allprokaryotic organisms, including those within all of the phyla in theKingdom Procaryotae. It is intended that the term encompass allmicroorganisms considered to be bacteria including Mycoplasma,Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms ofbacteria are included within this definition including cocci, bacilli,spirochetes, spheroplasts, protoplasts, etc. “Gram negative” and “grampositive” refer to staining patterns obtained with the Gram-stainingprocess which is well known in the art (See e.g., Finegold and Martin,Diagnostic Microbiology, 6th Ed. (1982), CV Mosby St. Louis, pp 13-15).

As used herein, the term “lipid membrane” refers to, in its broadestsense, a thin sheet or layer comprising lipid molecules. It is intendedthat the term encompass all “biomembranes” (i.e., any organic membraneincluding, but not limited to, plasma membranes, nuclear membranes,organelle membranes, and synthetic membranes). Typically, membranes arecomposed of lipids, proteins, glycolipids, steroids, sterol and/or othercomponents. As used herein, the term “membrane fragment” refers to anyportion or piece of a membrane.

As used herein, the term “lipid” refers to a variety of compounds thatare characterized by their solubility in organic solvents. Suchcompounds include, but are not limited to, fats, waxes, steroids,sterols, glycolipids, glycosphingolipids (including gangliosides),phospholipids, terpenes, fat-soluble vitamins, prostaglandins,carotenes, and chlorophylls. As used herein, the phrase “lipid-basedmaterials” refers to any material that contains lipids.

As used herein, the term “liposome” refers to artificially producedspherical lipid complexes that can be induced to segregate out ofaqueous media.

General Description of the Invention

The present invention relates to the fields of molecular biology,cellular biology, immunology, oncology, developmental biology, stem cellgrowth and differentiation, general laboratory science, andmicrobiology, and in particular to methods and compositions based onliquid crystal assays and other biophotonic assays for detecting andquantifying the presence of cells, cell secretory products includingpolypeptides and enzymes, microorganisms (including but not limited toviruses, bacteria, fungi and parasites) and particulate matter on asubstrate. The ability to correlate an output signal with cell numbermakes the devices of the present invention widely useful for assays ofcell adhesion as well as cell proliferation, cell death and cellulardifferentiation. Additionally, methods are described that allowquantification of movement of cells in response to cytoactive agents aswell as under control conditions. Compounds that promote cell migrationmay be chemotactic (e.g., compounds that stimulate directed cellmigration in response to a gradient) or chemokinetic (e.g., compoundsthat stimulate cell migration that is not gradient or directionallydependent) agents. Additionally, inhibition of cell migration may bequantified. It is contemplated that adhesion is indicative of a changein functionality of the cell. Indeed, adhesion represents a firstessential step in cell migration. Adhesion is also a requirement forsurvival and subsequent proliferation of anchorage dependent cell typessuch as fibroblasts and epithelial cells. For example, adhesiondocuments an essential change in leukocytes that participatesubsequently in diapedesis and is an essential component of woundhealing.

It is contemplated that proliferation is indicative of normal growthand/or replacement of effete cells in the maintenance of homeostasis.Proliferation is also a fundamental aspect of neoplasia and an essentialcomponent of wound healing, ontogeny, inflammation and the immuneresponse. Adhesion, migration, differentiation and proliferation arefundamental cell behaviors that are modulated by soluble factors (e.g.,cytokines, chemokines, neuropeptides, neurotrophins, polypeptide growthfactors) as well as by the extracellular matrix constituents (e.g.,collagens, laminin, vitronectin, fibronectin) and influenced by othercells and their products in the environment. Examining how theseprocesses are modulated in vitro provides insights into normalphysiologic processes, assists in elucidating the impact of factors inisolation and in combination with each other and allows dissection ofdisease processes such as neoplasia.

The present invention provides devices and methods for the determinationof cell number in combination with cell proliferation and cell adhesionassays. As such, the present invention provides a single platform tomultiple cell assays, including, but not limited to, adhesion,migration, proliferation, invasion, death, differentiation andcontraction assays. Therefore, the devices and methods of the presentinvention provide distinct advantages over and complement methodsincluding direct cell counting using microscopy and a hemocytometer orautomated cell counting devices (e.g., a Coulter counter); calorimetricassays that utilize substrate conversion by intracellular enzymes (e.g.,MTT assays); direct colorimetric assays based on extraction of dyes (andsubsequent quantification) after initial vital staining of cells;fluorometric assays based on enzymatic conversion (e.g., CalceinAM-molecular probes that provides a fluorometrically converted substratefor intracellular esterases; fluorometric assays based on DNA binding(e.g. Hoechst dyes); colorimetric or fluorometric assays based onidentification of intracellular correlative indicators of cellproliferation such as detection of Proliferating Cell Nuclear Antigen(PCNA); BRDU labeling of DNA and examining by microscopy; radiometricassays based on incorporation of tritiated thymidine; and flow cytometrywith propidium iodide labeling.

Accordingly, in some embodiments the invention contemplates theplacement of any analyte (particles, virus, bacteria, fungi, parasites,cells, proteins including enzymes) onto an ordered surface such thatliquid crystals placed on top are prevented from accessing the orderinginfluence of the underlying substrate. Thus, the presence of the analyteis revealed in a non-specific manner. In other embodiments, the presenceof an analyte will interfere with (disrupt) the ordering influence of anelectrical and/or magnetic field. In still other embodiments of theinvention, the presence of the analyte changes the structure of thesurface such that the ordering influence of the surface on the liquidcrystals is changed. In other embodiments, the spatial and orientationalorder of the bound analytes is of a type such that the presence of theanalyte introduces order into the liquid crystal.

The present invention further contemplates the use of liquid crystalfilms to report and amplify changes in the order of an underlyingsubstrate. While not limited to any particular mechanism, it is believedthat this phenomenon is a result of the introduction of order into amesogenic layer deposited onto a disordered substrate, or conversely,the introduction of disorder into a mesogenic layer deposited on anordered substrate. It is contemplated that the order (or disorder) in asubstrate is generated by the regular (or irregular) occurrence ofnanostructures, microstructures or molecules on the substrate.

An aligned (ordered) surface can be created using a wide variety oftechniques including oblique deposition of gold, simple rubbing ofpolymeric surfaces or by the rubbing of proteins covalently bound to avariety of surfaces, micromolding of polymers, micro/nanoabrasiveprocessing of surfaces, lithographic methods of fabrication, includingoptical lithography, electron beam lithography and x-ray lithography.Additionally, LCs can be ordered on isotropic surfaces that supportparticles that have been oriented or organized in an anisotropicfashion. Any biologic event that imparts order to a disordered surfaceor disorder to an ordered surface (including isotropic surfaces thatsupport particles that have been oriented or organized in an anisotropicfashion) is readily detected by the use of liquid crystals. In certainembodiments, the nanostructures are chemical moieties, including but notlimited to, polypeptides and proteins, nucleic acids, lipids,phospholipids, carbohydrates, ions, organic molecules and inorganicmolecules, and the like. In certain other embodiments, thenanostructures are physical features in the substrate surface producedby photolithography, electron beam lithography, micromolding, scanningprobe methods including atomic force microscopy and scanning tunnelingmicroscopy, photoetching, chemical etching, microcontact printing,chemical spotting, mechanical abrasives, high pressure water etching andthe like.

In preferred embodiments, the surface order is created by rubbing apolymeric surface or a surface that has had one or more proteins and/orother biological moieties (e.g., sugars, specific receptors or cellreceptor recognition sequences [e.g., RGD]) covalently bound to it. Inother embodiments, the surface is doped with submicron to 10 μm sizedparticles (particles can be anisometric and/or isometric depending onthe method used) and aligning or partially aligning them through the useof external fields (including but not limited to electric fields,magnetic fields, shear fields and/or fluid flow). The particles can alsobe delivered to the surface by microcontact printing the particles froma topographically textured stamp. The topography of the stamp can alignand order the particles prior to their transfer to the surface.

Accordingly, the present invention contemplates that the orientation ofmagnetic micro-nanoparticles can be manipulated (e.g., ordered) bycontrolled application of a magnetic field or electrical current. Insome embodiments, the nanostructures on a substrate surface are producedby a combination of the physical methods.

The present invention contemplates that the changes in the local order(or disorder) of the mesogenic layer result from physically perturbingthe layer. The change in order of the surface is readily observed withplacement of a thin layer of liquid crystal and the use of polarizers orspecific wavelengths of light and photodiode or a charge coupled device(CCD). The change in order of the surface could be either localized anddiscreet in nature or generalized.

In some embodiments, the perturbations are caused by the migration ofcells across the surface. In other embodiments, the perturbations arecaused by the adhesion, proliferation, morphological changes, lossand/or contraction of cells on the surface. In some of theseembodiments, the cell membrane is intact, while in other embodiments thecell membrane is solubilized. In some embodiments, where cellularfunctions (e.g., cellular motility, adhesion, proliferation, apoptosis)are being assayed, the secretion of cellular factors that alter (e.g.,adhere to) the surface may change local surface order. In someembodiments, liquid crystalline membranes are used to nonspecificallyreport biomechanical transduction events associated with cell adhesions,migration and contraction. It is contemplated that these assays functionby a change of order in the crystalline membrane itself. For example,one such contemplated assay material is a film of liquid crystal thatspontaneously adsorbs phospholipids at its surface. In anotherembodiment, an elastomeric liquid crystalline material is used. In yetanother embodiment, a polymer-stabilized or polymer-dispersed liquidcrystal is used.

Preferred embodiments of the present invention are directed to assaysfor quantitating the effects of chemotactic and chemokinetic agents aswell as inhibitors of cell migration on cells (e.g., cancer cells). Thepresent invention is not limited however to providing assays forquantitating the effects of agents suspected of being involved in cancerformation and metastasis on cellular functions and motility.

Many motility factors for cancer cells and non-malignant cells weredescribed first as being growth factors. A motility factor converts astatic, adherent cell to a motile status, a transition that ischaracterized by the appearance of membrane ruffling, lamellae,filopodia and pseudopodia. Several motility factors have been describedfor cancer cells including: (1) autocrine motility factor (AMF) whichstimulates chemokinesis and chemotaxis of metastatic melanoma cells inan autocrine fashion; (2) autotaxin; (3) scatter factor/hepatocytegrowth factor (e.g., ligands for the c-met oncogene product, a tyrosinekinase receptor family member); (4) TGF-α and EGF; (5) insulin-likegrowth factors; and (6) constituents of the extracellular matrix such asfibronectin; 7) PDGF; 8) LPA; 9) amphiregulin; and 10) chemokines. Thesefactors stimulate chemokinesis and chemotaxis. The present inventionspecifically contemplates assays for detecting and quantifying theeffects of one or more of these motility factors on cancer cell (andnon-cancer cell) motility.

While metastatic cancer cells are thought to rely upon the processes ofcell adhesion, deformability, motility, and receptor recognition forcreating metastases, none of these processes are unique to metastaticcancer cells. These processes have been observed in numerousnon-cancerous cell types and cellular processes (e.g., trophoblastimplantation, mammary gland involution, embryonic morphogenesis,hematopoietic stem cells, and tissue remodeling).

Thus, certain embodiments of the present invention are directed toassays for quantifying the effects of potential cytoactive agents (e.g.,mitogenic, growth inhibiting, chemotactic, and chemokinetic agents,inhibitors of cell migration, as well as agents that promote or inhibitcell adhesion, death, or differentiation) on cell types involved infertility and conception, stem cell differentiation and proliferation,gene therapy and cell targeting, immunology, and diseases characterizedby abnormal cell motility or migration. Certain other embodimentsprovide assays for quantitating the effects of cytoactive agents onbacteria, archaea, and eukarya. In certain embodiments the cytoactiveagent being assayed is an attractant (e.g., positive chemotactic agent)of one or more cell types. In certain other embodiments the agent is astimulant to cell migration but is non-directional in its effects (e.g.,a chemokinetic agent). In certain other embodiments the cytoactive agentis an inhibitor or repellent of one or more cell types. In someembodiments, and in particular those embodiments directed to assaysemploying bacteria and archaea cells, potential tactic agents include,but are not limited to, phototaxis, aerotaxis, or osmotaxis agents, andthe like.

The present invention also provides devices and methods for using liquidcrystals to determine the metabolic states of cells, for detectingsecretory products of cells, for analyzing the structure of cellcytoskeletons, and for analyzing and measuring cell invasion intomatrices. In particularly preferred embodiments, the present inventionprovides matrices that comprise a liquid crystal component and matricesthat orient liquid crystals. In other preferred embodiments, thesubstrate itself is a liquid crystal. Each of these embodiments isdescribed in more detail below. It will be recognized that many of theexemplary assay devices are not limited to use with liquid crystaldisplay methodology. In particular, many of the devices and methods areuseful with detection methodologies, including but not limited tofluorimetry, densitometry, colorimetry, and radiometry.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the fields of molecular biology,cellular biology, immunology, oncology, cellular differentiation,general laboratory science and microbiology, and in particular tomethods and compositions based on liquid crystal assays and otherbiophotonic assays for detecting and quantifying the presence of cellson a substrate, for the detection and quantification of cell secretoryproducts including enzymes and the quantification of fundamental cellbehaviors such as adhesion, differentiation, death, proliferation, andmigration. The cell assays can be performed under control conditions aswell as for evaluation of cell responsiveness to cytoactive agents.Liquid crystal-based assay systems (LC assays) are described in WO99/63329, which is herein incorporated by reference, and Gupta et al.,Science 279:2077-2080 (1998). See also Seung-Ryeol Kim, Rahul R. Shah,and Nicholas L. Abbott; Orientations of Liquid Crystals on MechanicallyRubbed Films of Bovine Serum Albumin: A Possible Substrate forBiomolecular Assays Based on Liquid Crystals, Analytical Chemistry;2000; 72(19); 4646-4653; Justin J. Skaife and Nicholas L. Abbott;Quantitative Interpretation of the Optical Textures of Liquid CrystalsCaused by Specific Binding of Immunoglobulins to Surface-Bound Antigens,Langmuir; 2000; 16(7);3529-3536; Vinay K. Gupta and Nicholas L. Abbott;Using proplets of Nematic Liquid Crystal To Probe the Microscopic andMesoscopic Structure of Organic Surfaces, Langmuir; 1999; 15(21);7213-7223. R. R. Shah and N. L. Abbott, Principles for measurement ofchemical exposure based on recognition-driven anchoring transitions inliquid crystals, Science; 2001; 293(5533):1296-99. WO 01/61357 describesthe detection of viruses using liquid crystal based assays. These assaysutilize a patterned detection region on a substrate that organizesmesogens in a homeotropic orientation. The assays are designed so thatbinding of a virus to the detection regions disrupts the homeotropicorientation.

The LC assays of the present invention are useful for detecting thepresence, spatial distribution, and states of cells, and also fordetecting and quantitating a wide variety of molecules (e.g., chemicaland biological materials, such as ions, proteins and polypeptides,lipids, polysaccharides, nucleic acids, low molecular weight compounds,and the like) that act as chemotactic or cellular adhesion andproliferation inducing agents or are secreted by cells in response toenvironmental stimuli. The LC assays may also be used to investigateregional differences on individual cell surfaces as to the expression ofsurface receptors or secretion of molecules such as growth factors,chemokines, cytokines, enzymes and constituents of the extracellularmatrix. The LC assays of the present inventions are also useful fordetecting and quantitating cells attached to a substrate (directlyapplicable to assays of cell adhesion, cell differentiation and cellproliferation) as well as assaying migration in a variety of cell types(e.g., cancer cells, lymphocytes, bacteria, archaea, etc.). This allowsthe quantitative analysis of the impact of a wide array of cytoactivecompounds that may promote, have no effect or inhibit the fundamentalcellular processes.

The assays can also be used to discern subtle changes in the motility ofa cell (or particular type of cell) upon contact with a suspectedcytoactive agent. Indeed, the assays can be used to detect and quantifya variety of biological and non-biological entities including, but notlimited to, eukaryotic cells, prokaryotic cells, viruses, bacteria,fungi, beads, and particles in suspension. LC assays of the presentinvention are used to directly detect interruption of their surfacesand, in preferred embodiments, the assayed materials do not requirelabels, fluorescent dyes, colored substrates, or secondary antibodies.

In some preferred embodiments, the present inventions finds use in thedetection and/or analysis of cells, including, but not limited toinclude, Chinese hamster ovary cells (CHO-K1, ATCC CCl-61); bovinemammary epithelial cells (ATCC CRL 10274; bovine mammary epithelialcells); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture; see, e.g., Graham et al., J. Gen Virol.,36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); mousesertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 [1980]); monkeykidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68[1982]); MRC 5 cells; FS4 cells; rat fibroblasts (208F cells); MDBKcells (bovine kidney cells); human hepatoma line (Hep G2), and, forexample, the following cancerous cells or cells isolated from thefollowing carcinomas: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, Ewing's tumor,lymphangioendotheliosarcoma, synovioma, mesothelioma, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilns' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrbm's macroglobulinemia, and heavychain disease.

Furthermore, the LC assays of the present invention are readilyadaptable to multi-array formats that permit simultaneous quantitationof the effects of one or more cytoactive agents upon one or more typesof target cells and appropriate controls. Adaptability to multi-arrayformats also makes the LC assays of the present invention useful inhigh-throughput screening applications such as drug discovery. The LCassays of the present invention are also fast because the liquidcrystals reorient in response to alterations in a surface in seconds.

In some embodiments of the present invention, the LC assays comprise asubstrate to which recognition moieties are attached, preferably via anorganic layer on the substrate (See, e.g., U.S. Pat. No. 6,284,197,which is incorporated herein by reference). In preferred embodiments,the substrate or organic layer serves to uniformly orient the liquidcrystal. In some preferred embodiments, the substrate surface isprepared by rubbing, micro/nanoblasting (i.e., abrasion of a surfacewith submicron particles to create roughness), high pressure wateretching or oblique deposition of a metal. In some embodiments thesubstrate consists of a protein coated surface. In some embodiments, thesubstrate provides a uniform, homogenous or planar surface, while inother embodiments, the surface is heterogeneous and/or containstopographic features. In some particularly preferred embodiments, thesubstrate is patterned to allow quantification.

Ordered nanostructured anisotropic surfaces introduce order into (e.g.,align) liquid crystal films that are placed onto their surfaces. Thisordering influence of the substrate can be eliminated by specificbinding of target molecules to receptors immobilized upon the surface inwhich the dimensions of the topographic features of the substrate arematched to that of the target molecule. Another method contemplatedherein for the detection and quantification of cells, microorganisms(e.g., bacteria, viruses, fungi, parasites) and particulate matter, isto simply block access to the ordering influence of the underlyingnanostructured substrate by placement of cells, microorganisms,particulate matter or non-specifically or specifically secreted ordeposited proteins or other matrices on its surface.

Accordingly, in some embodiments, assay substrates are provided thatfacilitate detection and quantification of cells, microorganisms andparticulate matter in a nonspecific manner. These assay substratespreferably include at least one assay region that orients liquidcrystals. In preferred embodiments, the assay region that orients liquidcrystals comprises a surface. As described above, the surface may haveintrinsic features (e.g., anisotropic structures) that orient liquidcrystals, or the surface may have associated therewith particles orother materials that orient liquid crystals (i.e., extrinsic orderingmaterials). These extrinsic ordering materials may align liquid crystalsdirectly or may be aligned by application of an extrinsic force such asa magnetic field, electric field, or polarized light. As such, thepresent invention is not limited to any particular method of providing asurface or other structure that orients liquid crystals. For example, insome embodiments, nanorods are deposited on the surface and alignedusing a motive force exemplified by but not limited to electric fields,magnetic fields, and fluid flow across surface.

In some preferred embodiments, the assays of the present invention maybe used to quantify the amount of particulate matter in a sample, forexample, the amount of viral particles in a sample. Currently, virusesare usually quantified by biological assays (e.g., LD50, TCID 50 orplaque titrations). Particle counts aren't often performed because theyrequire electron microscopy. The assays of the present invention may beused to quantify viral particles in a sample by applying the viralparticles to a substrate surface via diffusion of an electric field, andoverlaying the substrate with a liquid crystal. The disorder of theliquid crystal is related to the number of particles on the orderedsurface.

In some preferred embodiments, it is contemplated that when a particularcell type of interest is deposited upon, adheres to andior migratesacross the surface of the LC substrate, the topographic features incontact with the liquid crystal film are altered thus altering thealignment of the overlying LC film. In the case of a migrating cell,local order may be disrupted by its passage over an anisotropicallyordered surface or may locally introduce order into a randomly orderedsurface.

In preferred embodiments, the concentration of chemotactic agents andthe number of cells contacted to a LC assay surface are controlled suchthat distinct cellular migration paths are observable. According to thepresent invention, the disruption of orientation can be detected by avariety of methods, including viewing with polarized light, measuringthe threshold electrical field required to change the orientation of theliquid crystal, and viewing in the presence of dichroic agents.

In other preferred embodiments, one or more chemical agents and celltypes are contacted (e.g., arrayed) over the surface of a disorderedsurface such that subsequent cellular adhesion and migration causes theuniform orientation of the previously disordered liquid crystal alongthe discrete path of motility. The ordered liquid crystals can be viewedusing polarizing filters and/or using a specific wavelength orcombination of wavelengths of light. In some embodiments, the change inorder of the surface is localized and discreet in nature. In otherembodiments, the change in the order of the surface is generalized.

Accordingly, the present invention provides improved substrates anddevices for LC assays, including quantitative LC assays. Forconvenience, the description of the present invention is divided intothe following sections: I. Substrates; II. Organic Layers; III.Recognition Moieties; IV. Mesogenic Layers; V. Patterned LiquidCrystals; and VI. Analytical Devices.

I. Substrates

Substrates that are useful in practicing the present invention can bemade of practically any physicochemically stable material. In apreferred embodiment, the substrate material is non-reactive towards theconstituents of the mesogenic layer. The substrates can be either rigidor flexible and can be either optically transparent or optically opaque.The substrates can be electrical insulators, conductors orsemiconductors. Further, the substrates can be substantially impermeableto liquids, vapors and/or gases or, alternatively, the substrates can bepermeable to one or more of these classes of materials. Exemplarysubstrate materials include, but are not limited to, inorganic crystals,inorganic glasses, inorganic oxides, metals, organic polymers andcombinations thereof.

A. Inorganic Crystal and Glasses

In some embodiments of the present invention, inorganic crystals andinorganic glasses are utilized as substrate materials (e.g., LiF, NaF,NaCl, KBr, KI, CaF₂, MgF₂, HgF₂, BN, AsS₃, ZnS, Si₃N₄ and the like). Thecrystals and glasses can be prepared by art standard techniques (See,e.g., Goodman, C. H. L., Crystal Growth Theory and Techniques, PlenumPress, New York 1974). Alternatively, the crystals can be purchasedcommercially (e.g., Fischer Scientific). The crystals can be the solecomponent of the substrate or they can be coated with one or moreadditional substrate components. Thus, it is within the scope of thepresent invention to utilize crystals coated with, for example one ormore metal films or a metal film and an organic polymer. Additionally, acrystal can constitute a portion of a substrate which contacts anotherportion of the substrate made of a different material, or a differentphysical form (e.g., a glass) of the same material. Other usefulsubstrate configurations utilizing inorganic crystals and/or glasseswill be apparent to those of skill in the art.

B. Inorganic Oxides

In other embodiments of the present invention, inorganic oxides areutilized as the substrate. Inorganic oxides of use in the presentinvention include, for example, Cs₂O, Mg(OH)₂, TiO₂, ZrO₂, CeO₂, Y₂O₃,Cr₂O₃, Fe₂O₃, NiO, ZnO, Al₂O₃, SiO₂ (glass), quartz, In₂O₃, SnO₂, PbO₂and the like. The inorganic oxides can be utilized in a variety ofphysical forms such as films, supported powders, glasses, crystals andthe like. A substrate can consist of a single inorganic oxide or acomposite of more than one inorganic oxide. For example, a composite ofinorganic oxides can have a layered structure (i.e., a second oxidedeposited on a first oxide) or two or more oxides can be arranged in acontiguous non-layered structure. In addition, one or more oxides can beadmixed as particles of various sizes and deposited on a support such asa glass or metal sheet. Further, a layer of one or more inorganic oxidescan be intercalated between two other substrate layers (e.g.,metal-oxide-metal, metal-oxide-crystal).

In a presently preferred embodiment, the substrate is a rigid structurethat is impermeable to liquids and gases. In this embodiment, thesubstrate consists of a glass plate onto which a metal, such as gold islayered by evaporative deposition. In a still further preferredembodiment, the substrate is a glass plate (SiO₂) onto which a firstmetal layer such as titanium has been layered. A layer of a second metalsuch as gold is then layered on top of the first metal layer.

C. Metals

In still further embodiments of the present invention, metals areutilized as substrates. The metal can be used as a crystal, a sheet or apowder. The metal can be deposited onto a backing by any method known tothose of skill in the art including, but not limited to, evaporativedeposition, sputtering, electroless deposition, electrolytic depositionand adsorption or deposition of preformed particles of the metalincluding metallic nanoparticles.

Any metal that is chemically inert towards the mesogenic layer will beuseful as a substrate in the present invention. Metals that are reactiveor interactive towards the mesogenic layer will also be useful in thepresent invention. Metals that are presently preferred as substratesinclude, but are not limited to, gold, silver, platinum, palladium,nickel and copper. In one embodiment, more than one metal is used. Themore than one metal can be present as an alloy or they can be formedinto a layered “sandwich” structure, or they can be laterally adjacentto one another. In a preferred embodiment, the metal used for thesubstrate is gold. In a particularly preferred embodiment the metal usedis gold layered on titanium.

The metal layers can be either permeable or impermeable to materialssuch as liquids, solutions, vapors and gases.

D. Organic Polymers

In still other embodiments of the present invention, organic polymersare utilized as substrate materials. Organic polymers useful assubstrates in the present invention include polymers that are permeableto gases, liquids and molecules in solution. Other useful polymers arethose that are impermeable to one or more of these same classes ofcompounds.

Organic polymers that form useful substrates include, for example,polyalkenes (e.g., polyethylene, polyisobutene, polybutadiene),polyacrylics (e.g., polyacrylate, polymethyl methacrylate,polycyanoacrylate), polyvinyls (e.g., polyvinyl alcohol, polyvinylacetate, polyvinyl butyral, polyvinyl chloride), polystyrenes,polycarbonates, polyesters, polyurethanes, polyamides, polyimides,polysulfone, polysiloxanes, polyheterocycles, cellulose derivative(e.g., methyl cellulose, cellulose acetate, nitrocellulose),polysilanes, fluorinated polymers, epoxies, polyethers and phenolicresins (See, Cognard, J. ALIGNMENT OF NEMATIC LIQUID CRYSTALS AND THEIRMIXTURES, in Mol. Cryst. Liq. Cryst. 1:1-74 (1982)). Presently preferredorganic polymers include polydimethylsiloxane, polyethylene,polyacrylonitrile, cellulosic materials, polycarbonates and polyvinylpyridinium.

In a presently preferred embodiment, the substrate is permeable and itconsists of a layer of gold, or gold over titanium, which is depositedon a polymeric membrane, or other material, that is permeable toliquids, vapors and/or gases. The liquids and gases can be purecompounds (e.g., chloroform, carbon monoxide) or they can be compoundsthat are dispersed in other molecules (e.g., aqueous protein solutions,herbicides in air, alcoholic solutions of small organic molecules).Useful permeable membranes include, but are not limited to, flexiblecellulosic materials (e.g., regenerated cellulose dialysis membranes),rigid cellulosic materials (e.g., cellulose ester dialysis membranes),rigid polyvinylidene fluoride membranes, polydimethylsiloxane and tracketched polycarbonate membranes.

In a further preferred embodiment, the layer of gold on the permeablemembrane is itself permeable. In a still further preferred embodiment,the permeable gold layer has a thickness of about 70 Angstroms or less.

In those embodiments wherein the permeability of the substrate is not aconcern and a layer of a metal film is used, the film can be as thick asis necessary for a particular application. For example, if the film isused as an electrode, the film can be thicker than in an embodiment inwhich it is necessary for the film to be transparent or semi-transparentto light.

Thus, in a preferred embodiment, the film is of a thickness of about0.01 nanometer to about 1 micrometer. In a further preferred embodiment,the film is of a thickness of about 5 nanometers to about 100nanometers. In yet a further preferred embodiment, the film is of athickness of about 10 nanometers to about 50 nanometers.

E. Substrate Surfaces

It is contemplated that the nature of the surface of the substrate has aprofound effect on the anchoring of the mesogenic layer that isassociated with the surface. The surface can be engineered by the use ofmechanical and/or chemical techniques. The surface of each of the aboveenumerated substrates can be substantially smooth (planar).Alternatively, the surface can be roughened or patterned by rubbing,etching, grooving, stretching, stressing, impacting, nanoblasting,oblique deposition or other similar techniques known to those with skillin the art. Additionally, an ordered surface can be created by thedeposition (decoration) of nano-micro sized particles that are depositedonto a planar surface. These particles may be placed onto the surface inan ordered array using a nanostamper or “negative” nanostamper (seeFIGS. 1A and 1B) or may be placed on in a random array and subsequentlyordered using motive forces exemplified by but not limited to electricfields, magnetic fields and fluid flow. Of particular relevance is thetexture of the surface that is in contact with the mesogenic compounds.

Thus, in one preferred embodiment, the substrate is glass or an organicpolymer and the surface has been prepared by rubbing. Rubbing can beaccomplished using virtually any material including tissues, paper,fabrics, brushes, polishing paste, etc. In a preferred embodiment, therubbing is accomplished by use of a diamond rubbing paste. In anotherpreferred embodiment, the face of the substrate that contacts themesogenic compounds is a metal layer that has been obliquely depositedby evaporation. In a further preferred embodiment, the metal layer is agold layer.

In other embodiments of the present invention, anisotropic surfaces areprepared by nanoblasting a substrate with nanometer scale beads (e.g.,1-200 nm, preferably 50-100 nm) at a defined angle of incidence (e.g.,from about 5-85 degrees, preferably about 45 degrees). The nanoblastedsurface can be utilized as is or can be further modified, such as byobliquely depositing gold on the surface.

In still further embodiments, the ansiotropic surfaces of the devices ofthe present invention are prepared by stretching an appropriatesubstrate. For example, polymers substrates such as polystyrene can bestretched by heating to a temperature above the glass transitiontemperature of the substrate, applying a tensile force, and cooling to atemperature below the glass transition temperature before removing theforce.

In some embodiments, the present invention provides substrates withheterogenous features for use in the various devices and methods. Insome embodiments, the heterogenity is a uniform or non-uniform gradientin topography across the surface. For example, gold can be depositedonto a substrate at varying angles of incidence. Regions containing golddeposited at a near-normal angle of incidence will cause non-uniformanchoring of the liquid crystal, while areas in which the angle ofincidence was greater than 10 degrees will uniformly orient crystals.Alternatively, the heterogeneity may be the presence of two or moredistinct scales of topography distributed uniformly across thesubstrate. It is contemplated that such substrates are useful forincreasing the dynamic range of detection of analytes or for detectingthe presence of analytes of a different size within a sample.

The substrate can also be patterned using techniques such asphotolithography (Kieinfield et al., J. Neurosci. 8:4098-120 (1998)),photoetching, chemical etching, microcontact printing (Kumar et al.,Langmuir 10: 1498-511 (1994)), and chemical spotting.

The size and complexity of the pattern on the substrate is limited onlyby the resolution of the technique utilized and the purpose for whichthe pattern is intended. For example, using microcontact printing,features as small as 200 nm have been layered onto a substrate (See,Xia, Y.; Whitesides, G., J. Am. Chem. Soc. 117:3274-75 (1995)).Similarly, using photolithography, patterns with features as small as 1μm have been produced (See, Hickman et al., J. Vac. Sci. Technol.12:607-16 (1994)). Patterns which are useful in the present inventioninclude those which comprise features such as wells, enclosures,partitions, recesses, inlets, outlets, channels, troughs, diffractiongratings and the like.

In a presently preferred embodiment, the patterning is used to produce asubstrate having a plurality of adjacent wells, wherein each of thewells is isolated from the other wells by a raised wall or partition andthe wells do not fluidically communicate. Thus, an analyte (e.g., apotential chemotactic agent), or other substance, placed in a particularwell remains substantially confined to that well. In another preferredembodiment, the patterning allows the creation of channels through thedevice whereby an agent can enter and/or exit the device.

The pattern can be printed directly onto the substrate or,alternatively, a “lift off” technique can be utilized. In the lift offtechnique, a patterned resist is laid onto the substrate, an organiclayer is laid down in those areas not covered by the resist and theresist is subsequently removed. Resists appropriate for use with thesubstrates of the present invention are known to those of skill in theart (See, e.g., Kleinfield et al., J. Neurosci. 8:4098-120 (1998)).Following removal of the photoresist, a second organic layer, having astructure different from the first organic layer, can be bonded to thesubstrate on those areas initially covered by the resist. Using thistechnique, substrates with patterns having regions of different chemicalcharacteristics can be produced. Thus, for example, a pattern having anarray of adjacent wells can be created by varying thehydrophobicity/hydrophilicity, charge and other chemical characteristicsof the pattern constituents. In one embodiment, hydrophilic compoundscan be confined to individual wells by patterning walls usinghydrophobic materials. Similarly, positively or negatively chargedcompounds can be confined to wells having walls made of compounds withcharges similar to those of the confined compounds. Similar substrateconfigurations are accessible through microprinting a layer with thedesired characteristics directly onto the substrate (See, Mrkish, M.;Whitesides, G. M., Ann. Rev. Biophys. Biomol. Struct. 25:55-78 (1996)).

In yet another preferred embodiment, the patterned substrate controlsthe anchoring alignment of the liquid crystal. In a particularlypreferred embodiment, the substrate is patterned with an organiccompound that forms a self-assembled monolayer. In this embodiment, theorganic layer controls the azimuthal orientation and/or polarorientation of a supported mesogenic layer.

In still further preferred embodiments, the present invention providessurfaces that have particles (preferably nano- to micro-sized particlesdistributed thereon). It is contemplated that such particles find useboth in orienting liquid crystals and in masking underlying orientingfeatures associated with the surface, depending on the configuration. Insome embodiments, a stamping device is utilized that deposits micro- tonano-sized particles on a surface (See, e.g., FIG. 1A). It is possibleto make a stamp from relatively friable materials that are transferredto the substrate upon mechanical contact with the substrate. Examples ofsuch transferable materials include but are not limited to charcoal,chalk, soapstone, graphite, pumice and other easily fragmented andtransferred materials. This can include a synthetic laminated material,prepared such that fracturing layers are designed into the material.

It is also possible to create an aligned surface using mechanicaltransfer of organized or aligned particles (“positive transfer stamp”).Such a device would have a design similar to FIG. 1A but instead of thearrayed ridges being composed of a friable material, they are composedof a non-consumable material appropriate for picking up particles anddepositing them on a test surface in an ordered array. For example, ahydrophobic stamp containing topography may be fabricated. Theparticles, when deposited onto the topography are either organized oraligned such that when transferred to a substrate on which the cells aregrown, the liquid crystal is aligned. These particles can be hidden,displaced or reoriented when cells are deposited onto, attach to, growor migrate on the surface. In some preferred embodiments of a transferstamp, electric or magnetic fields are applied across the stamp tocollect particles. In further preferred embodiments, the applied fieldis changed so as to affect the transfer of particles to the testsubstrate in an ordered array. The ordered array of particles wouldsubsequently align mesogens in a LC film placed on the test substrate'ssurface. In some embodiments of the invention, the positions of theparticles once transferred to the substrates are made tolerant tohandling and unintended disruption of ordering by chemical or physicalattachment to the substrate.

FIG. 2A depicts the use of a negative nanostamping device. In thisembodiment, nano to micro-sized particulate matter is randomlydistributed across the test surface of a substrate and anisotropic ordercreated by using a “negative stamping” method that removes material inan ordered fashion leaving an anisotropically patterned surface thataligns the mesogens in an LC film.

In some embodiments, the substrate surface further comprises a pluralityof magnetic particles (e.g., metallic nanorods) evenly distributedacross the substrate surface. In certain of these embodiments, themagnetic particles are salted onto the surface of the substrate. Thepresent invention contemplates that the orientation of magneticnanoparticles can be manipulated (e.g., ordered) by controlledapplication of a magnetic field or electrical current.

In some embodiments, the cells, microorganisms, or particles on a planarsurface are visualized by introducing order into the liquid crystal filmby methods exemplified by, but not limited to, the use of electricfields, magnetic fields and/or the placement of nano- and/ormicroparticles deposited on the surface that are ordered on initialdeposition (using a nano or microstamper device) or ordered subsequentto deposition by use of electric fields, magnetic fields or the use offluid flow. In the case of ordering the LC film with electric ormagnetic fields or by fluid flow without the use of an intrinsicanisotropic nanostructured substrate or the use of anisotropicallyordered nano and/or microparticles decorating the surface, the orderintroduced in the LC film by these methods is disrupted locally by thepresence of cells, microorganisms and/or particulate matter as well asspecifically immobilized target molecules. The degree of disruption isquantifiable by the magnitude of the ordering motive force (e.g.electric field) required to maintain a uniformly ordered LC film despitethe presence of a disruptive element represented by the presence ofmaterial (e.g., cells, microorganisms, particulate matter) on thesurface.

Additionally, it is contemplated that upon cessation of application ofsaid motive force the cells, microorganisms, and/or particulate matterpresent on the surface introduce disorder into the LC layer proportionalto their size and density. In some embodiments, the analytes (e.g.,particulate matter, cells, or microorganisms) are passively placed ontothe surface and nonspecifically associated with the surface; while inother embodiments, the analytes are specifically immobilized onto (e.g.,target molecules immobilized by specific binding to receptors) theplanar surface. This principle is illustrated in FIGS. 2A, 2B, and 3. InFIG. 2A it can be seen that the mesogens are randomly arranged aroundthe binding sequence immobilized on the planar surface (100) with itstarget analyte (300). The element to be detected on the planar surfacecould also be a particle, a cell or a microorganism (bacteria, fungi,virus, parasite). These elements to be detected may be specificallybound to the surface as depicted or could simply be resting on thesurface or non-specifically (i.e. no specific binding sequence present)associated to the surface. On a planar surface with or without elementsto be detected the mesogens have a random distribution (200).

Referring to FIG. 2B, an electric field (400) is used as an example ofthe motive force that could be used to align the mesogens in the liquidcrystal, though other motive forces that align liquid crystal mesogens(exemplified by, but not limited to magnetic fields and fluid flow)could also be used. It can be appreciated that the motive force issufficient to overcome the introduction of disorder into the mesogeniclayer by the presence of elements on the planar surface. While the forceis applied, all mesogenic elements are aligned (200). Referring to FIG.3, the effect of greatly reducing or discontinuing application of analigning motive force is illustrated. The mesogenic elements away fromthe target analyte, cell, microorganism or particle on the surface stayaligned. The mesogenic elements in association with the target analyte,cell, microorganism or particle have disorder introduced into them. Thisdisorder is communicated many molecular lengths away and allowsdetection using polarized light or specific wavelengths or combinationsof wavelengths of light. It will be appreciated that the orientation ofthe liquid crystal on the substrate, in the absence of the applied fieldand bound analyte, has a degenerate degree of orientational freedom.This means that the liquid crystal can be irreversibly changed by theapplication of the field. An example of such an interface is aliquid-liquid interface (e.g., degenerate azimuthal orientations), butthe invention is not confined to operation at this interface. Examplesof degenerate anchoring of liquid crystals on solid surfaces are wellknown to those skilled in the art (see Physics of Liquid Crystals, Prostand de Gennes).

Additionally, in other embodiments, the present invention contemplatesmethods in which a planar surface is seeded with cells, microorganismsor particulate matter. A liquid crystal film is placed onto the surfaceand alignment is introduced using a motive force (e.g., electric fields,magnetic fields or fluid flow). The present invention is not limited toany particular mechanism of action. Indeed, an understanding of themechanism of action is not necessary to practice the present invention.Nevertheless, the magnitude of the force required to maintain alignmentof the liquid crystal is proportional to the degree of disruptive forcepossessed by an element on the surface. A major contributor to thedisruptive force (i.e., the ability to introduce disorder into themesogenic layer) is related to its size. Collecting information (e.g., acurve) of the change in light transmission as a function of motive forceapplied necessary to maintain a uniformly aligned LC film providesquantitative data on the relative amount of different sized particles.FIG. 4 demonstrates that the force/light transmission curves differsignificantly between two populations of elements placed on a planarsurface.

In FIG. 4, the solid line depicts the force curve associated with apopulation of analytes having heterogeneously sized particles with a fewpredominant sizes and the dashed line is consistent with the presence ofa homogenous population of relatively large particles. The positions ofthese curves also depend on the number of particles on the surface. Fora given applied force (e.g., electric field), the transmission of lightis greater if more particles are present on the surface. That is, theparticles prevent the uniform alignment promoted by the applied field.

Additionally, rather than using a nanostructured or microstructuredsubstrate, it is possible to use an electric field or magnetic field toorder the LC or enhance the optical contrast between regions of thesubstrate occupied or not occupied by cells or other particulate matter,microorganisms (bacteria, fungi, viruses, parasites) as well as specificor non-specific analytes. These embodiments are described in more detailbelow.

F. Liquid Crystal Substrates and Matrices

Additionally, in some embodiments, liquid crystalline substrates areemployed to report biomechanical forces imparted to the substrate by theadhesion, movement and contraction of cells. In some embodiments, the LCsubstrate is functionalized by the adhesion of matrix proteins, glass orother biocompatible polymeric substrates to support cell function. Inother embodiments, the LC substrate comprises a low molecular weight LCfilm spread over a solid substrate, a LC gel that is formed by particlesor gelator molecules dispersed in the liquid crystal, a polymeric liquidcrystal, a polymer-stabilized liquid crystalline film that is preparedby the polymerization of a network of polymer in a liquid crystal(similar to that used in polymer-dispersed liquid crystal displays), orpolymer stabilized liquid crystals (e.g., lyotropic liquid crystals).The surfaces of the liquid crystal films can be functionalized byimmobilization of receptors that are anchored to the surface by one ofthe various methods of immobilization known to those skilled in the art,including but not limited to covalent attachment, physisorption forexample by using a receptor coupled to a surface-active molecule, or byuse of polymers adsorbed to the surface of the liquid crystallinesubstrate.

In some preferred embodiments, a hybrid liquid crystalline film isprepared from a combination of a liquid crystal and extracellular matrix(ECM) constituents or a combination of liquid crystals and cell adhesionmolecules (e.g. ICAM, selecting, syndeeans). In another preferredembodiment of the invention, the extracellular matrix constituents orsynthetic mimics of ECM exhibits liquid crystalline order that isaltered by the present of cells attached to the matrix. The degree ofdisorder or order of the liquid crystal film can be assessed andquantified using white light or using a specific wavelength orcombination of wavelengths of light.

In yet other embodiments, the use of a liquid crystalline matrix isemployed that reports the transduction of biomechanical forces fromcells placed on the surface into the matrix that alters the passage oflight. Additionally, in some embodiments, thin films of extracellularmatrices (e.g., collagen or fibronectin), cell surface adhesionmolecules, thin polymeric films that support cell function and may havebeen functionalized by the immobilization of extracellular matrixconstituents or specific binding sequences (e.g., RGD) that promote cellfunction, and hybrid extracellular-liquid crystalline matrices are usedto report biomechanical forces imparted into these films by cellularprocesses such as adhesion, migration or contraction.

In still further embodiments, matrices (e.g., ECMs) are provided thathave been modified to orient liquid crystals. Such matrices also finduse in adhesion, migration, and invasion assays.

II. Organic Layers

In addition to the ability of a substrate to anchor a mesogenic layer,an organic layer attached to the substrate is similarly able to providesuch anchoring. A wide range of organic layers can be used inconjunction with the present invention. These include, but are notlimited to, organic layers formed from organosulfur compounds (includingthiols and disulfides), organosilanes, amphiphilic molecules,cyclodextrins, polyols (e.g., poly(ethyleneglycol),poly(propyleneglycol), fullerenes, and biomolecules (e.g., proteins,lipids, nucleic acids, polysaccharides, phospholipids and the like). Inpreferred embodiments that employ organic layers, the layer is selectedafter considering the affects the layer will have on the assay. Forexample, assays where the migration of cells are being considered, oneskilled in the art would select an organic layer that is not expected tointerfere with reactions' conditions and cellular migration,alternatively, one skilled in the art could select an organic layerexpected to either promote or retard cellular migration.

A. Anchoring

An organic layer that is bound to, supported on or adsorbed onto, thesurface of the substrate can anchor a mesogenic layer. As used herein,the term “anchoring” refers to the set of orientations adopted by themolecules in the mesogenic phase. The mesogenic layer will adoptparticular orientations while minimizing the free energy of theinterface between the organic layer and the mesogenic layer. Theorientation of the mesogenic layer is referred to as an “anchoringdirection.” A number of anchoring directions are possible.

It is contemplated that the particular anchoring direction adopted willdepend upon the nature of the mesogenic layer, the organic layer and thesubstrate. Anchoring directions of use in the present invention include,for example, conical anchoring, degenerate anchoring, homeotropicanchoring, multistable anchoring, planar anchoring and tilted anchoring.Planar anchoring and homeotropic anchoring are preferred with planaranchoring being most preferred.

The anchoring of mesogenic compounds by surfaces has been extensivelystudied for a large number of systems (See, for example, Jerome, Rep.Prog. Phys. 54:391-451 (1991)). The anchoring of a mesogenic substanceby a surface is specified, in general, by the orientation of thedirector of the bulk phase of the mesogenic layer. The orientation ofthe director, relative to the surface, is described by a polar angle(measured from the normal of the surface) and an azimuthal angle(measured in the plane of the surface).

Control of the anchoring of mesogens has been largely based on the useof organic surfaces prepared by coating surface-active molecules orpolymer films on inorganic (e.g., silicon oxide) substrates followed bysurface treatments such as rubbing. Other systems which have been founduseful include surfaces prepared through the reactions of organosilaneswith various substrates (See, for example, Yang et al, InMICROCHEMISTRY: SPECTROSCOPY AND CHEMISTRY IN SMALL DOMAINS; Masuhara etal., Eds.; North-Holland, Amsterdam, 1994; p. 441).

Molecularly designed surfaces formed by organic layers on a substratecan be used to control both the azimuthal and polar orientations of asupported mesogenic layer. Self-assembled monolayers (SAMs) can bepatterned on a surface. For example, patterned organic layers made fromCH₃(CH₂)₁₄SH and CH₃(CH₂)₁₅SH on obliquely deposited gold produce asupported mesogenic layer which is twisted 90°. Other anchoring modesare readily accessible by varying the chain length and the number ofspecies of the organic layer constituents (See, Gupta and Abbott,Science 276:1533-1536 (1997)).

Transitions between anchoring modes have been obtained on a series oforganic layers by varying the structure of the organic layer. Structuralfeatures that have been found to affect the anchoring of mesogeniccompounds include, for example, the density of molecules within theorganic layer, the size and shape of the molecules constituting theorganic layer and the number of individual layers making up the bulkorganic layer.

The density of the organic layer on the substrate has been shown to havean effect on the mode of mesogen anchoring. For example, transitionsbetween homeotropic and degenerate anchorings have been obtained onsurfactant monolayers by varying the density of the monolayers (See,Proust et al., Solid State Commun. 11:1227-30 (1972)). Thus, it iswithin the scope of the present invention to tailor the anchoring modeof a mesogen by controlling the density of the organic layer on thesubstrate.

The molecular structure, size and shape of the individual moleculesmaking up the organic layer also affect the anchoring mode. For example,it has been demonstrated that varying the length of the aliphatic chainsof surfactants on a substrate can also induce anchoring transitions;with long chains, a homeotropic anchoring is obtained while with shortchains, a conical anchoring is obtained with the tilt angle θ increasingas the chain becomes shorter (See, e.g., Porte, J. Physique 37:1245-52(1976)). Additionally, recent reports have demonstrated that the polarangle of the mesogenic phase can be controlled by the choice of theconstituents of the organic layer. See, Gupta and Abbott, Langmuir12:2587-2593 (1996). Thus, it is within the scope of the presentinvention to engineer the magnitude of the anchoring shift as well asthe type of shift by the judicious choice of organic layer constituents.

Biomolecules can also be used as organic layers. (see Seung-Ryeol Kim,Rahul R. Shah, and Nicholas L. Abbott; Orientations of Liquid Crystalson Mechanically Rubbed Films of Bovine Serum Albumin: A PossibleSubstrate for Biomolecular Assays Based on Liquid Crystals, AnalyticalChemistry; 2000; 72(19); 4646-4653.). A preferred embodiment when usingbiomolecules as organic layers is based on the mechanical rubbing of theorganic layer with a fabric cloth following chemical immobilization ofthe organic layer on the surface of a substrate.

A wide variety of organic layers are useful in practicing the presentinvention. These organic layers can comprise monolayers, bilayers andmultilayers. Furthermore, the organic layers can be attached by covalentbonds, ionic bonds, physisorption, chemisorption and the like,including, but not limited to, hydrophobic interactions, hydrophilicinteractions, van der Waals interactions and the like.

In a presently preferred embodiment, organic layers which formself-assembled monolayers are used. The use of self-assembled monolayers(SAMs) formed from alkanethiols on thin, semitransparent films of goldin studies on the anchoring of liquid crystals on surfaces has beenreported (See, Drawhorn and Abbott, J. Phys. Chem. 45:16511 (1995)). Theprincipal result of that work was the demonstration that SAMs formedfrom n-alkanethiols with long (CH₃(CH₂)₁₅SH) and short (CH₃(CH₂)₄SH orCH₃(CH₂)₉SH) aliphatic chains can homeotropically anchor mesogens. Incontrast, single-component SAMs caused non-uniform, planar, or tiltedanchoring at room temperature.

In the discussion that follows, self-assembled monolayers are utilizedas an exemplary organic layer. This use is not intended to be limiting.It will be understood that the various configurations of theself-assembled monolayers and their methods of synthesis, bindingproperties and other characteristics are equally applicable to each ofthe organic layers of use in the present invention.

B. Self-Assembled Monolayers

Self-assembled monolayers are generally depicted as an assembly oforganized, closely packed linear molecules. There are two widely-usedmethods to deposit molecular monolayers on solid substrates:Langmuir-Blodgett transfer and self-assembly. Additional methods includetechniques such as depositing a vapor of the monolayer precursor onto asubstrate surface and the layer-by-layer deposition of polymers andpolyelectrolytes from solution (Guy Ladam, Pierre Schaaf, Frédéric J. G.Cuisinier, Gero Decher, and Jean-Claude Voegel; Protein Adsorption ontoAuto-Assembled Polyelectrolyte Films, Langmuir; 2001; 17(3); 878-882).

The composition of a layer of a SAM useful in the present invention canbe varied over a wide range of compound structures and molar ratios. Inone embodiment, the SAM is formed from only one compound. In a presentlypreferred embodiment, the SAM is formed from two or more components. Inanother preferred embodiment, when two or more components are used, onecomponent is a long-chain hydrocarbon having a chain length of between10 and 25 carbons and a second component is a short-chain hydrocarbonhaving a chain length of between 1 and 9 carbon atoms. In particularlypreferred embodiments, the SAM is formed from CH₃(CH₂)₁₅SH andCH₃(CH₂)₄SH or CH₃(CH₂)₁₅SH and CH₃(CH₂)₉SH. In any of the abovedescribed embodiments, the carbon chains can be functionalized at theω-terminus (e.g., NH₂, COOH, OH, CN), at internal positions of the chain(e.g., aza, oxa, thia) or at both the ω-terminus and internal positionsof the chain.

The mesogenic layer can be layered on top of one SAM layer or it can besandwiched between two SAM layers. In those embodiments in which themesogenic layer is sandwiched between two SAMs, a second substrate,optionally substantially identical in composition to that bearing theSAM can be layered on top of the mesogenic layer. Alternatively acompositionally different substrate can be layered on top of themesogenic layer. In a preferred embodiment, the second substrate ispermeable. In yet another preferred embodiment two substrates are used,but only one of the substrates has an attached organic layer.

When the mesogenic layer is sandwiched between two layers of SAMsseveral compositional permutations of the layers of SAMs are available.For example, in one embodiment, the first organic layer and the secondorganic layer have substantially identical compositions and both of theorganic layers bear an attached recognition moiety. A variation on thisembodiment utilizes first and second organic layers with substantiallysimilar compositions, wherein only one of the layers bears a recognitionmoiety.

In another embodiment, the first and second organic layers havesubstantially different compositions and only one of the organic layershas an attached recognition moiety. In a further embodiment, the firstorganic layer and said second organic layer have substantially differentcompositions and both of the organic layers have an attached recognitionmoiety.

In a presently preferred embodiment, the organic layers havesubstantially identical compositions and one or both of the organiclayers have attached thereto a recognition moiety.

A recognition moiety can be attached to the surface of a SAM by any of alarge number of art-known attachment methods. In one preferredembodiment, a reactive SAM component is attached to the substrate andthe recognition moiety is subsequently bound to the SAM component viathe reactive group on the component and a group of complementaryreactivity on the recognition moiety (See, e.g., Hegner et al. Biophys.J. 70:2052-2066 (1996)). In another preferred embodiment, therecognition moiety is attached to the SAM component prior toimmobilizing the SAM component on the substrate surface: the recognitionmoiety-SAM component cassette is then attached to the substrate. In astill further preferred embodiment, the recognition moiety is attachedto the substrate via a displacement reaction. In this embodiment, theSAM is preformed and then a fraction of the SAM components are displacedby a recognition moiety or a SAM component bearing a recognition moiety.

C. Functionalized SAMs

The discussion that follows focuses on the attachment of a reactive SAMcomponent to the substrate surface. This focus is for convenience onlyand one of skill in the art will understand that the discussion isequally applicable to embodiments in which the SAM component-recognitionmoiety is preformed prior to its attachment to the substrate. As usedherein, “reactive SAM components” refers to components that have afunctional group available for reaction with a recognition moiety orother species following the attachment of the component to thesubstrate.

Currently favored classes of reactions available with reactive SAMcomponents are those that proceed under relatively mild conditions.These include, but are not limited to nucleophilic substitutions (e.g.,reactions of amines and alcohols with acyl halides), electrophilicsubstitutions (e.g., enamine reactions) and additions to carbon-carbonand carbon-heteroatom multiple bonds (e.g., Michael reaction,Diels-Alder addition). These and other useful reactions are discussed inMarch, ADVANCED ORGANIC CHEMISTRY, Third Ed., John Wiley & Sons, NewYork, 1985.

According to the present invention, a substrate's surface isfunctionalized with SAM, components and other species by covalentlybinding a reactive SAM component to the substrate surface in such a wayas to derivatize the substrate surface with a plurality of availablereactive functional groups. Reactive groups which can be used inpracticing the present invention include, for example, amines, hydroxylgroups, carboxylic acids, carboxylic acid derivatives, alkenes,sulfhydryls, siloxanes, etc.

A wide variety of reaction types are available for the functionalizationof a substrate surface. For example, substrates constructed of a plasticsuch as polypropylene, can be surface derivatized by chromic acidoxidation, and subsequently converted to hydroxylated or aminomethylatedsurfaces. Substrates made from highly crosslinked divinylbenzene can besurface derivatized by chloromethylation and subsequent functional groupmanipulation. Additionally, functionalized substrates can be made frometched, reduced polytetrafluoroethylene.

When the substrates are constructed of a siliaceous material such asglass, the surface can be derivatized by reacting the surface Si—OH,SiO—H, and/or Si—Si groups with a functionalizing reagent. When thesubstrate is made of a metal film, the surface can be derivatized with amaterial displaying avidity for that metal.

In a preferred embodiment, wherein the substrates are made from glass,the covalent bonding of the reactive group to the glass surface isachieved by conversion of groups on the substrate's surface by a siliconmodifying reagent such as:(RO)₃—Si—R¹—X¹  (1)where R is an alkyl group, such as methyl or ethyl, R¹ is a linkinggroup between silicon and X and X is a reactive group or a protectedreactive group. The reactive group can also be a recognition moiety asdiscussed below. Silane derivatives having halogens or other leavinggroups beside the displayed alkoxy groups are also useful in the presentinvention.

A number of siloxane functionalizing reagents can be used, for example:

-   -   1. Hydroxyalkyl siloxanes (Silylate surface, functionalize with        diborane, and H₂0₂ to oxidize the alcohol)        -   a. allyl trichlorosilane→→3-hydroxypropyl        -   b. 7-oct-1-enyl trichlorosilane→→8-hydroxyoctyl    -   2. Diol (dihydroxyalkyl) siloxanes (silylate surface and        hydrolyze to diol)        -   a. (glycidyl            trimethoxysilane→→(2,3-dihydroxypropyloxy)propyl    -   3. Aminoalkyl siloxanes (amines requiring no intermediate        functionalizing step).        -   a. 3-aminopropyl trimethoxysilane→aminopropyl    -   4. Dimeric secondary aminoalkyl siloxanes        -   a. bis(3-trimethoxysilylpropyl)            amine→bis(silyloxylpropyl)amine.

It will be apparent to those of skill in the art that an array ofsimilarly useful functionalizing chemistries is available when SAMcomponents other than siloxanes are used. Thus, for example, similarlyfunctionalized alkyl thiols can be attached to metal films andsubsequently reacted to produce the functional groups such as thoseexemplified above.

In another preferred embodiment, the substrate is at least partially ametal film, such as a gold film, and the reactive group is tethered tothe metal surface by an agent displaying avidity for that surface. In apresently preferred embodiment, the substrate is at least partially agold film and the group which reacts with the metal surface comprises athiol, sulfide or disulfide such as:Y—S—R²—X²  (2)R² is a linking group between sulfur and X² and X² is a reactive groupor a protected reactive group. X² can also be a recognition moiety asdiscussed below. Y is a member selected from the group consisting of H,R³ and R³—S—, wherein R² and R³ are independently selected. When R² andR³ are the same, symmetrical sulfides and disulfides result, and whenthey are different, asymmetrical sulfides and disulfides result.

A large number of functionalized thiols, sulfides and disulfides arecommercially available (Aldrich Chemical Co., St. Louis). Additionally,those of skill in the art have available to them a manifold of syntheticroutes with which to produce additional such molecules. For example,amine-functionalized thiols can be produced from the correspondinghalo-amines, halo-carboxylic acids, etc. by reaction of these haloprecursors with sodium sulfhydride. See, e.g., Reid, ORGANIC CHEMISTRYof BIVALENT SULFUR, VOL 1, pp. 21-29, 32-35, vol. 5, pp. 27-34, ChemicalPublishing Co., New York, 1.958, 1963. Additionally, functionalizedsulfides can be prepared via alkylthio-de-halogenation with a mercaptansalt (See, Reid, ORGANIC CHEMISTRY OF BIVALENT SULFUR, vol. 2, pp.16-21, 24-29, vol. 3, pp. 11-14, Chemical Publishing Co., New York,1960). Other methods for producing compounds useful in practicing thepresent invention will be apparent to those of skill in the art.

In another preferred embodiment, the functionalizing reagent providesfor more than one reactive group per each reagent molecule. Usingreagents such as Compound 3, below, each reactive site on the substratesurface is, in essence, “amplified” to two or more functional groups:(RO)₃—Si—R²—(X²)_(n)  (3)where R is an alkyl group, such as methyl, R² is a linking group betweensilicon and X², X² is a reactive group or a protected reactive group andn is an integer between 2 and 50, and more preferably between 2 and 20.

Similar amplifying molecules are also of use in those embodimentswherein the substrate is at least partially a metal film. In theseembodiments the group which reacts with the metal surface comprises athiol, sulfide or disulfide such as in Formula (4):Y—S—R²—(X²)_(n)  (4)As discussed above, R² is a linking group between sulfur and X² and X²is a reactive group or a protected reactive group. X² can also be arecognition moiety. Y is a member selected from the group consisting ofH, R³ and R³—S—, wherein R² and R³ are independently selected.

R groups of use for R¹, R² and R³ in the above described embodiments ofthe present invention include, but are not limited to, alkyl,substituted alkyl, aryl, arylalkyl, substituted aryl, substitutedarylalkyl, acyl, halogen, hydroxy, amino, alkylamino, acylamino, alkoxy,acyloxy, aryloxy, aryloxyalkyl, mercapto, saturated cyclic hydrocarbon,unsaturated cyclic hydrocarbon, heteroaryl, heteroarylalkyl, substitutedheteroaryl, substituted heteroarylalkyl, heterocyclic, substitutedheterocyclic and heterocyclicalkyl groups.

In each of Formulae 1-4, above, each of R¹, R² and R³ are either stableor they can be cleaved by chemical or photochemical reactions. Forexample, R groups comprising ester or disulfide bonds can be cleaved byhydrolysis and reduction, respectively. Also within the scope of thepresent invention is the use of R groups that are cleaved by light suchas, for example, nitrobenzyl derivatives, phenacyl groups, benzoinesters, etc. Other such cleaveable groups are well-known to those ofskill in the art.

In another preferred embodiment, the organosulfur compound is partiallyor entirely halogenated. An example of compounds useful in thisembodiment includes:X¹Q₂C(CQ¹ ₂)_(m)Z¹(CQ² ₂)_(n)SH  (5)wherein, X¹ is a member selected from the group consisting of H, halogenreactive groups and protected reactive groups. Reactive groups can alsobe recognition moieties as discussed below. Q, Q¹ and Q² areindependently members selected from the group consisting of H andhalogen. Z¹ is a member selected from the group consisting of —CQ₂-,—CQ¹ ₂-, —CQ² ₂-, —O—, —S—, NR⁴—, —C(O)NR⁴ and R⁴NC(O0-, in which R⁴ isa member selected from the group consisting of H, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl and heterocyclic groups and mand n are independently a number between 0 and 40.

In yet another preferred embodiment, the organic layer comprises acompound according to Formula 5 above, in which Q, Q¹ and Q² areindependently members selected from the group consisting of H andfluorine. In a still further preferred embodiment, the organic layercomprises compounds having a structure according to Formulae (6) and(7):CF₃(CF₂)_(m)Z¹(CH₂)_(n)SH  (6)CF₃(CF₂)_(o)Z²(CH₂)_(p)SH  (7)wherein, Z¹ and Z² are members independently selected from the groupconsisting of —CH₂—, —O—, —S—, NR⁴, —C(O)NR⁴ and R⁴NC(O)— in which R⁴ isa member selected from the group consisting of H, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl and heterocyclic groups. In apresently preferred embodiment, the Z groups of adjacent moleculesparticipate in either an attractive (e.g., hydrogen bonding) orrepulsive (e.g., van der Waals) interaction.

In Formulae 6 and 7, m is a number between 0 and 40, n is a numberbetween 0 and 40, o is a number between 0 and 40 and p is a numberbetween 0 and 40.

In a further preferred embodiment, the compounds of Formulae 6 and 7 areused in conjunction with an organosulfur compound, either halogenated orunhalogenated, that bears a recognition moiety.

When the organic layer is formed from a halogenated organosulfurcompound, the organic layer can comprise a single halogenated compoundor more than one halogenated compound having different structures.Additionally, these layers can comprise a non-halogenated organosulfurcompound.

The reactive functional groups (X¹ and X²) are, for example:

(a) carboxyl groups and various derivatives thereof including, but notlimited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters,acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,alkenyl, alkynyl and aromatic esters;

(b) hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.

(c) haloalkyl groups wherein the halide can be later displaced with anucleophilic group such as, for example, an amine, a carboxylate anion,thiol anion, carbanion, or an alkoxide ion, thereby resulting in thecovalent attachment of a new group at the site of the halogen atom;

(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido groups;

(e) aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, forexample, to form sulfonamides;

(g) thiol groups which can be converted to disulfides or reacted withacyl halides;

(h) amine or sulfhydryl groups which can be, for example, acylated oralkylated;

(i) alkenes which can undergo, for example, cycloadditions, acylation,Michael addition, etc; and

(j) epoxides which can react with, for example, amines and hydroxylcompounds. The reactive moieties can also be recognition moieties. Thenature of these groups is discussed in greater detail below.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reaction controlling theattachment of the functionalized SAM component onto the substrate'ssurface. Alternatively, the reactive functional group can be protectedfrom participating in the reaction by the presence of a protectinggroup. Those of skill in the art will understand how to protect aparticular functional group from interfering with a chosen set ofreaction conditions. For examples of useful protecting groups, seeGreene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley &Sons, New York, 1991.

In a preferred embodiment, the SAM component bearing the recognitionmoiety is attached directly and essentially irreversibly via a “stablebond” to the surface of the substrate. A “stable bond”, as used herein,is a bond that maintains its chemical integrity over a wide range ofconditions (e.g., amide, carbamate, carbon-carbon, ether, etc.). Inanother preferred embodiment, the SAM component bearing the recognitionmoiety is attached to the substrate surface by a “cleaveable bond”. A“cleaveable bond”, as used herein, is a bond that is designed to undergoscission under conditions which do not degrade other bonds in therecognition moiety-analyte complex. Cleaveable bonds include, but arenot limited to, disulfide, imine, carbonate and ester bonds.

In certain embodiments, it is advantageous to have the recognitionmoiety attached to a SAM component having a structure that is differentthan that of the constituents of the bulk SAM. In this embodiment, thegroup to which the recognition moiety is bound is referred to as a“spacer arm” or “spacer.” Using such spacer arms, the properties of theSAM adjacent to the recognition moiety can be controlled. Propertiesthat are usefully controlled include, for example, hydrophobicity,hydrophilicity, surface-activity and the distance of the recognitionmoiety from the plane of the substrate and/or the SAM. For example, in aSAM composed of alkanethiols, the recognition moiety can be attached tothe substrate or the surface of the SAM via an amine terminatedpoly(ethyleneglycol). Numerous other combinations of spacer arms andSAMs are accessible to those of skill in the art.

The hydrophilicity of the substrate surface can be enhanced by reactionwith polar molecules such as amine-, hydroxyl- and polyhydroxylcontaining molecules. Representative examples include, but are notlimited to, polylysine, polyethyleneimine, poly(ethyleneglycol) andpoly(propyleneglycol). Suitable functionalization chemistries andstrategies for these compounds are known in the art (See, for example,Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACSSymposium Series Vol. 469, American Chemical Society, Washington, D.C.1991).

The hydrophobicity of the substrate surface can be modulated by using ahydrophobic spacer arm such as, for example, long chain diamines, longchain thiols, α, ω-amino acids, etc. Representative hydrophobic spacersinclude, but are not limited to, 1,6-hexanediamine, 1,8-octanediamine,6-aminohexanoic acid and 8-aminooctanoic acid.

The substrate surface can also be made surface-active by attaching tothe substrate surface a spacer that has surfactant properties. Compoundsuseful for this purpose include, for example, aminated or hydroxylateddetergent molecules such as, for example, 1-aminododecanoic acid.

In another embodiment, the spacer serves to distance the recognitionmoiety from the substrate or SAM. Spacers with this characteristic haveseveral uses. For example, a recognition moiety held too closely to thesubstrate or SAM surface may not react with incoming analyte, or it mayreact unacceptably slowly. When an analyte is itself stericallydemanding, the reaction leading to recognition moiety-analyte complexformation can be undesirably slowed, or not occur at all, due to themonolithic substrate hindering the approach of the two components.

In another embodiment, the physicochemical characteristics (e.g.,hydrophobicity, hydrophilicity, surface activity, conformation) of thesubstrate surface and/or SAM are altered by attaching a monovalentmoiety which is different in composition than the constituents of thebulk SAM and which does not bear a recognition moiety. As used herein,“monovalent moiety” refers to organic molecules with only one reactivefunctional group. This functional group attaches the molecule to thesubstrate. “Monovalent moieties” are to be contrasted with thebifunctional “spacer” groups described above. Such monovalent groups areused to modify the hydrophilicity, hydrophobicity, bindingcharacteristics, etc. of the substrate surface. Examples of groupsuseful for this purpose include long chain alcohols, amines, fattyacids, fatty acid derivatives, poly(ethyleneglycol) monomethyl ethers,etc.

When two or more structurally distinct moieties are used as componentsof the SAMs, the components can be contacted with the substrate as amixture of SAM components or, alternatively, the components can be addedindividually. In those embodiments in which the SAM components are addedas a mixture, the mole ratio of a mixture of the components in solutionresults in the same ratio in the mixed SAM. Depending on the manner inwhich the SAM is assembled, the two components do not phase segregateinto islands (See, Bain and Whitesides, J. Am. Chem. Soc. 111:7164(1989)). This feature of SAMs can be used to immobilize recognitionmoieties or bulky modifying groups in such a manner that certaininteractions, such as steric hindrance, between these molecules areminimized.

The individual components of the SAMs can also be bound to the substratein a sequential manner. Thus, in one embodiment, a first SAM componentis attached to the substrate's surface by “underlabeling” the surfacefunctional groups with less than a stoichiometric equivalent of thefirst component. The first component can be a SAM component liked to aterminal reactive group or recognition group, a spacer arm or amonovalent moiety. Subsequently, the second component is contacted withthe substrate. This second component can either be added instoichiometric equivalence, stoichiometric excess or can again be usedto underlabel to leave sites open for a third component.

III. Recognition Moieties

In some embodiments of the present invention, a “recognition moiety”attached to or associated with the substrate is utilized to bind to orotherwise interact with another molecule or molecules (e.g., analytes)or a cell. For example, in some embodiments, recognition moieties areattached to either ω-functionalized spacer arms or co-functionalized SAMcomponents that are in turn attached to or associated with thesubstrate. Furthermore, a recognition moiety can be presented by apolymer surface (e.g., a rubbed polymer surface).

In some preferred embodiments, the recognition moiety comprises anorganic functional group. In presently preferred embodiments, theorganic functional group is a member selected from the group consistingof amines, carboxylic acids, drugs, chelating agents, crown ethers,cyclodextrins or a combination thereof.

In another preferred embodiment, the recognition moiety is abiomolecule. In still further preferred embodiments, the biomolecule isa polypeptide or protein (e.g., specific receptors or cell receptorrecognition sequences [e.g., RGD]), antigen binding protein, peptide,nucleic acid (e.g., single nucleotides or nucleosides, oligonucleotides,polynucleotides and single- and higher-stranded nucleic acids), lipids,phospholipids, or a combination thereof. In a presently preferredembodiment, the recognition moiety is biotin. In some embodiments of thepresent invention, the recognition moiety is an antigen binding protein.Such antigen binding proteins include, but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments, and Fabexpression libraries. In particularly preferred embodiments, the proteinand polypeptide recognition moieties comprise proteins and peptidesequences normally found in the extracellular matrix that support cellattachment and function (e.g., collagens including types I, II, III andIV, laminins, fibronectin, vitronectin). In some embodiments, peptidesequences are attached as short functional sequences (e.g., RGD) or thefunctional sequences may be contained within longer peptide sequences(e.g., x-RGD-x). Examples of peptide sequences include, but are notlimited to, all of the known integrin binding sequences, including RGD,EILDV, LDV, LDVP, IDAP, PHSRN, SLDVP, and IDSP.

Various procedures known in the art may be used for the production ofpolyclonal antibodies. For the production of antibody, various hostanimals, including but not limited to rabbits, mice, rats, sheep, goats,etc., can be immunized by injection with the peptide corresponding to anepitope. In a preferred embodiment, the peptide is conjugated to animmunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels (e.g., aluminum hydroxide), surface activesubstances (e.g., lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (Bacille Calmette-Guerin)and Corynebacterium parvum).

For preparation of monoclonal antibodies, it is contemplated that anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture will find use with the presentinvention (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Theseinclude but are not limited to the hybridoma technique originallydeveloped by Köhler and Milstein (Köhler and Milstein, Nature256:495-497 [1975]), as well as the trioma technique, the human B-cellhybridoma technique (See e.g., Kozbor et al., Immunol. Tod., 4:72[1983]), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96 [1985]).

In addition, it is contemplated that techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; hereinincorporated by reference) will find use in producing specific singlechain antibodies that serve as recognition moieties. Furthermore, it iscontemplated that any technique suitable for producing antibodyfragments will find use in generating antibody fragments that are usefulrecognition moieties. For example, such fragments include but are notlimited to: F(ab′)2 fragment that can be produced by pepsin digestion ofthe antibody molecule; Fab′ fragments that can be generated by reducingthe disulfide bridges of the F(ab′)2 fragment, and Fab fragments thatcan be generated by treating the antibody molecule with papain and areducing agent. In still further embodiments, the recognition moietycomprises a phage displaying an antigen binding protein.

In some embodiments where the recognition moiety is a polynucleotide orpolypeptide, a plurality of recognition moieties are arrayed on thesubstrates using photo activated chemistry, microcontact printing, andink-jet printing. In particularly preferred embodiments,photolithography is utilized (See e.g., U.S. Pat. Nos. 6,045,996;5,925,525; and 5,858,659; each of which is herein incorporated byreference). Using a series of photolithographic masks to definesubstrate exposure sites, followed by specific chemical synthesis steps,the process constructs high-density arrays of oligonucleotides, witheach probe in a predefined position in the array. Multiple probe arraysare synthesized simultaneously on, for example, a large glass wafer. Thewafers are then diced, and individual probe arrays are packaged ininjection-molded plastic cartridges, which protect them from theenvironment and serve as chambers for hybridization.

In other embodiments, nucleic acid recognition moieties areelectronically captured on a suitable substrate (See e.g., U.S. Pat.Nos. 6,017,696; 6,068,818; and 6,051,380; each of which are hereinincorporated by reference). Through the use of microelectronics, thistechnology enables the active movement and concentration of chargedmolecules to and from designated test sites on its semiconductormicrochip. DNA capture probes unique to a given target areelectronically placed at, or “addressed” to, specific sites on themicrochip. Since DNA has a strong negative charge, it can beelectronically moved to an area of positive charge.

In still further embodiments, recognition moieties are arrayed on asuitable substrate by utilizing differences in surface tension (Seee.g., U.S. Pat. Nos. 6,001,311; 5,985,551; and 5,474,796; each of whichis herein incorporated by reference). This technology is based on thefact that fluids can be segregated on a flat surface by differences insurface tension that have been imparted by chemical coatings. Once sosegregated, oligonucleotide probes are synthesized directly on the chipby ink-jet printing of reagents. The array with its reaction sitesdefined by surface tension is mounted on an X/Y translation stage undera set of four piezoelectric nozzles, one for each of the four standardDNA bases. The translation stage moves along each of the rows of thearray and the appropriate reagent is delivered to each of the reactionsite. For example, the A amidite is delivered only to the sites whereamidite A is to be coupled during that synthesis step and so on. Commonreagents and washes are delivered by flooding the entire surface andthen removing them by spinning.

In still further embodiments, recognition moieties are spotted onto asuitable substrate. Such spotting can be done by hand with a capillarytube or micropipette, or by an automated spotting apparatus such asthose available from Affymetrix and Gilson (See e.g., U.S. Pat. Nos.5,601,980; 6,242,266; 6,040,193; and 5,700,637; each of which isincorporated herein by reference).

When the recognition moiety is an amine, in preferred embodiments, therecognition moiety will interact with a structure on the analyte thatreacts by binding to the amine (e.g., carbonyl groups, alkylhalogroups). In another preferred embodiment, the amine is protonated by anacidic moiety on the analyte of interest (e.g., carboxylic acid,sulfonic acid).

In certain preferred embodiments, when the recognition moiety is acarboxylic acid, the recognition moiety will interact with the analyteby complexation (e.g., metal ions). In still other preferredembodiments, the carboxylic acid will protonate a basic group on theanalyte (e.g. amine).

In another preferred embodiment, the recognition moiety is a drugmoiety. The drug moieties can be agents already accepted for clinicaluse or they can be drugs whose use is experimental, or whose activity ormechanism of action is under investigation. The drug moieties can have aproven action in a given disease state or can be only hypothesized toshow desirable action in a given disease state. In a preferredembodiment, the drug moieties are compounds that are being screened fortheir ability to interact with an analyte of choice. As such, drugmoieties that are useful in practicing the instant invention includedrugs from a broad range of drug classes having a variety ofpharmacological activities.

Classes of useful agents include, for example, non-steroidalanti-inflammatory drugs (NSAIDS). The NSAIDS can, for example, beselected from the following categories: (e.g., propionic acidderivatives, acetic acid derivatives, fenamic acid derivatives,biphenylcarboxylic acid derivatives and oxicams); steroidalanti-inflammatory drugs including hydrocortisone and the like;antihistaminic drugs (e.g., chlorpheniranune, triprolidine); antitussivedrugs (e.g., dextromethorphan, codeine, carmiphen and carbetapentane);antipruritic drugs (e.g., methidilizine and trimeprizine);anticholinergic drugs (e.g., scopolamine, atropine, homatropine,levodopa); anti-emetic and antinauseant drugs (e.g., cyclizine,meclizine, chlorpromazine, buclizine); anorexic drugs (e.g.,benzphetamine, phentermine, chlorphentermine, fenfluramine); centralstimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamineand methylphenidate); antiarrhythmic drugs (e.g., propanolol,procainamide, disopyraminde, quinidine, encamide); P-adrenergic blockerdrugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol);cardiotonic drugs (e.g., milrinone, aminone and dobutamine);antihypertensive drugs (e.g., enalapril, clonidine, hydralazine,minoxidil, guanadrel, guanethidine); diuretic drugs (e.g., amiloride andhydrochlorothiazide); vasodilator drugs (e.g., diltazem, amiodarone,isosuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs(e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs(e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine,bupivacaine, chlorprocaine, dibucaine); antidepressant drugs (e.g.,imipramine, desipramine, amitryptiline, nortryptiline); tranquilizer andsedative drugs (e.g., chlordiazepoxide, benacytyzine, benzquinamide,flurazapam, hydroxyzine, loxapine and promazine); antipsychotic drugs(e.g., chlorprothixene, fluphenazine, haloperidol, molindone,thioridazine and trifluoperazine); antimicrobial drugs (antibacterial,antifungal, antiprotozoal and antiviral drugs).

Antimicrobial drugs which are preferred for incorporation into thepresent composition include, for example, pharmaceutically acceptablesalts of β-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin,tetracycline, erythromycin, amikacin, triclosan, doxycycline,capreomycin, chlorhexidine, chlortetracycline, oxytetracycline,clindamycin, ethambutol, hexamidine isothionate, metronidazole;pentamidine, gentamycin, kanamycin, lineomycin, methacycline,methenamine, minocycline, neomycin, netilmycin, paromomycin,streptomycin, tobramycin, miconazole, and amanfadine, anti-microbialpeptides including but not limited to alpha and beta defensins,magainins, cecropins, bactenecins and indolicidin.

Other drug moieties of use in practicing the present invention includeantineoplastic drugs (e.g., antiandrogens (e.g., leuprolide orflutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol,cyclophosphamide, busulfan, cisplatin, a-2-interferon) anti-estrogens(e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate,mercaptopurine, thioguanine).

The recognition moiety can also comprise hormones (e.g.,medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide orsomatostatin, thyrotropin releasing hormone, angiotensin II, other smallpeptide hormones, as well as phospholipic hormones such asLysophosphatidic acid, platelet activating factor and eicosanoids);muscle relaxant drugs (e.g., cinnamedrine, cyclobenzaprine, flavoxate,orphenadrine, papaverine, mebeverine, idaverine, ritodrine,dephenoxylate, dantrolene and azumolen); antispasmodic drugs;bone-active drugs (e.g., diphosphonate and phosphonoalkylphosphinatedrug compounds); endocrine modulating drugs (e.g., contraceptives (e.g.,ethinodiol, ethinyl estradiol, norethindrone, mestranol, desogestrel,medroxyprogesterone), modulators of diabetes (e.g., glyburide orchlorpropamide), anabolics, such as testolactone or stanozolol,androgens (e.g., methyltestosterone, testosterone or fluoxymesterone),antidiuretics (e.g., desmopressin) and calcitonins).

Also of use in the present invention are estrogens (e.g.,diethylstilbesterol), glucocorticoids (e.g., triamcinolone,betamethasone, etc.) and progenstogens, such as norethindrone,ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g.,liothyronine or levothyroxine) or anti-thyroid agents (e.g.,methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormonesuppressors (e.g., danazol or goserelin), oxytocics (e.g.,methylergonovine or oxytocin) and prostaglandins, such as mioprostol,alprostadil or dinoprostone, can also be employed.

Other useful recognition moieties include immunomodulating drugs (e.g.,antihistamines, mast cell stabilizers, such as lodoxamide and/orcromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone,dexamethasone, prednisolone, methylprednisolone, beclomethasone, orclobetasol), histamine H₂ antagonists (e.g., famotidine, cimetidine,ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.Groups with anti-inflammatory activity, such as sulindac, etodolac,ketoprofen and ketorolac, are also of use. Other drugs of use inconjunction with the present invention will be apparent to those ofskill in the art.

When the recognition moiety is a chelating agent, crown ether orcyclodextrin, host-guest chemistry will dominate the interaction betweenthe recognition moiety and the analyte. The use of host-guest chemistryallows a great degree of recognition-moiety-analyte specificity to beengineered into a device of the invention. The use of these compounds tobind to specific compounds is well known to those of skill in the art.See, for example, Pitt et al. “The Design of Chelating Agents for theTreatment of Iron Overload,” In, INORGANIC CHEMISTRY IN BIOLOGY ANDMEDICINE; Martell, A. E., Ed.; American Chemical Society, Washington,D.C., 1980, pp. 279-312; Lindoy, L. F., THE CHEMISTRY OF MACROCYCLICLIGAND COMPLEXES; Cambridge University Press, Cambridge, 1989; Dugas,H., BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, andreferences contained therein.

Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, R. E., Whitaker, 1.R., Eds., American ChemicalSociety, Washington, D.C., 1982, pp. 370-387; Kasina et al. BioconjugateChem. 9:108-117 (1998); Song et al., Bioconjugate Chem. 8:249-255(1997).

In a presently preferred embodiment, the recognition moiety is apolyaminocarboxylate chelating agent such as ethylenediaminetetraaceticacid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). Theserecognition moieties can be attached to any amine-terminated componentof a SAM or a spacer arm, for example, by utilizing the commerciallyavailable dianhydride (Aldrich Chemical Co., Milwaukee, Wis.).

In still further preferred embodiments, the recognition moiety is abiomolecule such as a protein, nucleic acid, phospholipid fatty acidderivative, peptide or an antibody. Biomolecules useful in practicingthe present invention can be derived from any source. The biomoleculescan be isolated from natural sources or can be produced by syntheticmethods. Proteins can be natural proteins or mutated proteins. Mutationscan be effected by chemical mutagenesis, site-directed mutagenesis orother means of inducing mutations known to those of skill in the art.Proteins useful in practicing the instant invention include, forexample, enzymes, antigens, antibodies, structural proteins,transcription factors and receptors. Antibodies can be either polyclonalor monoclonal. Peptides, lipids, carbohydrates and nucleic acids can beisolated from natural sources or can be wholly or partially synthetic inorigin.

In those embodiments wherein the recognition moiety is a protein orantibody, the protein can be tethered to a SAM component or a spacer armby any reactive peptide residue available on the surface of the protein.In preferred embodiments, the reactive groups are amines orcarboxylates. In particularly preferred embodiments, the reactive groupsare the e-amine groups of lysine residues. Furthermore, these moleculescan be adsorbed onto the surface of the substrate or SAM by non-specificinteractions (e.g., chemisorption, physisorption).

Recognition moieties that are antibodies can be used to recognizeanalytes that are proteins, peptides, nucleic acids, lipids,phospholipids, lipopolysaccharides, saccharides or small molecules suchas drugs, herbicides, pesticides, infectious agents, industrialchemicals and agents of war. Methods of raising antibodies for specificmolecules are well-known to those of skill in the art. See, U.S. Pat.Nos. 5,147,786; 5,334,528; 5,686,237; 5,573,922; each of which isincorporated herein by reference. Methods for attaching antibodies tosurfaces are also art-known (See, Delamarche et al. Langmuir12:1944-1946 (1996)).

Peptides and nucleic acids can be attached to a SAM component or spacerarm. Both naturally-derived and synthetic peptides and nucleic acids areof use in conjunction with the present invention. These molecules can beattached to a SAM component or spacer arm by any available reactivegroup. For example, peptides can be attached through an amine, carboxyl,sulfhydryl, or hydroxyl group. Such a group can reside at a peptideterminus or at a site internal to the peptide chain. Nucleic acids canbe attached through a reactive group on a base (e.g., exocyclic amine)or an available hydroxyl group on a sugar moiety (e.g., 3′- or5′-hydroxyl). Polysaccharides, phospholipids, fatty acid derivatives andother lipids can be attached via hydroxyl groups on the sugar moiety orthe lipid portion as well as via free carboxyl or amino groups. Thepeptide and nucleic acid chains can be further derivatized at one ormore sites to allow for the attachment of appropriate reactive groupsonto the chain (See, Chrisey et al. Nucleic Acids Res. 24:3031-3039(1996)).

When the peptide or nucleic acid is a fully or partially syntheticmolecule, a reactive group or masked reactive group can be incorporatedduring the process of the synthesis. Many derivatized monomersappropriate for reactive group incorporation in both peptides andnucleic acids are know to those of skill in the art (See, for example,THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY, Vol. 2: “Special Methods inPeptide Synthesis,” Gross, E. and Melenhofer, J., Eds., Academic Press,New York (1980)). Many useful monomers are commercially available(Bachem, Sigma, etc.). This masked group can then be unmasked followingthe synthesis, at which time it becomes available for reaction with aSAM component or a spacer arm.

In other preferred embodiments, the peptide is attached directly to thesubstrate (See, Frey et al. Anal. Chem. 68:3187-3193 (1996)). In aparticularly preferred embodiment, the peptide is attached to a goldsubstrate through a sulfhydryl group on a cysteine residue. In anotherpreferred embodiment, the peptide is attached through a thiol to aspacer arm that terminates in, for example, an iodoacetamide,chloroacetamide, benzyl iodide, benzyl bromide, alkyl iodide or alkylbromide. Similar immobilization techniques are known to those of skillin the art (See, for example, Zull et al. J. Ind Microbiol. 13:137-143(1994)).

In another preferred embodiment, the recognition moiety forms aninclusion complex with the analyte of interest. In a particularlypreferred embodiment, the recognition moiety is a cyclodextrin ormodified cyclodextrin. Cyclodextrins are a group of cyclicoligosaccharides produced by numerous microorganisms. Cyclodextrins havea ring structure that has a basket-like shape. This shape allowscyclodextrins to include many kinds of molecules into their internalcavity (See, for example, Szejtli, J., CYCLODEXTRINS AND THEIR INCLUSIONCOMPLEXES; Akademiai Klado, Budapest, 1982; and Bender et al.,CYCLODEXTRIN CHEMISTRY, Springer-Verlag, Berlin, 1978).

Cyclodextrins are able to form inclusion complexes with an array oforganic molecules including, for example, drugs, pesticides, herbicidesand agents of war (See, Tenjarla et al., J. Pharm. Sci. 87:425-429(1998); Zughul et al., Pharm. Dev. Technol. 3:43-53 (1998); and Alberset al., Crit. Rev. Ther. Drug Carrier Syst. 12:311-337 (1995)).Importantly, cyclodextrins are able to discriminate between enantiomersof compounds in their inclusion complexes. Thus, in one preferredembodiment, the invention provides for the detection of a particularenantiomer in a mixture of enantiomers (See, Koppenhoefer et al. J.Chromatogr. A 793:153-164 (1998)).

The cyclodextrin recognition moiety can be attached to a SAM component,through a spacer arm or directly to the substrate (See, Yamamoto et al.,J. Phys. Chem. B 101:6855-6860 (1997)). Methods to attach cyclodextrinsto other molecules are well known to those of skill in thechromatographic and pharmaceutical arts (See, Sreenivasan, Appl. Polym.Sci. 60:2245-2249 (1996)).

IV. Mesogenic Layer

Any compound or mixture of compounds that forms a mesogenic layer can beused in conjunction with the present invention. The mesogens can formthermotropic or lyotropic liquid crystals. The mesogenic layer can beeither continuous or it can be patterned.

Both the thermotropic and lyotropic liquid crystals can exist in anumber of forms including nematic, chiral nematic, smectic, polarsmectic, chiral smectic, frustrated phases and discotic phases.

TABLE 1 Molecular structure of mesogens suitable for use in LiquidCrystal Assay Devices Mesogen Structure Anisaldazine

NCB

CBOOA

Comp A

Comp B

DB₇NO₂

DOBAMBC

nOm n = 1, m = 4: MBBA n = 2, m = 4: EBBA

nOBA n = 8: OOBA n = 9: NOBA

nmOBC

nOCB

nOSI

98P

PAA

PYP906

nSm

Presently preferred mesogens are displayed in Table 1. In a particularlypreferred embodiment, the mesogen is a member selected from the groupconsisting of 4-cyano-4′-pentylbiphenyl,N-(4-methoxybenzylidene)-4-butlyaniline and combinations thereof.

The mesogenic layer can be a substantially pure compound, or it cancontain other compounds that enhance or alter characteristics of themesogen. Thus, in one preferred embodiment, the mesogenic layer furthercomprises a second compound, for example an alkane, which expands thetemperature range over which the nematic and isotropic phases exist. Useof devices having mesogenic layers of this composition allows fordetection of the analyte recognition moiety interaction over a greatertemperature range.

In some preferred embodiments, the mesogenic layer further comprises adichroic dye or fluorescent compound. Examples of dichroic dyes andfluorescent compounds useful in the present invention include, but arenot limited to, azobenzene, BTBP, polyazocompounds, anthraquinone,perylene dyes, and the like. In particularly preferred embodiments, adichroic dye of fluorescent compound is selected that complements theorientation dependence of the liquid crystal so that polarized light isnot required to read the assay. In some preferred embodiments, if theabsorbance of the liquid crystal is in the visible range, then changesin orientation can be observed using ambient light without crossedpolars. In other preferred embodiments, the dichroic dye or fluorescentcompound is used in combination with a fluorimeter and the changes influorescence are used to detect changes in orientation of the liquidcrystal.

In another preferred embodiment, the analyte first interacts with therecognition moiety and the mesogenic layer is introduced in itsisotropic phase. The mesogenic layer is subsequently cooled to form theliquid crystalline phase. The presence of the analyte within regions ofthe mesogenic layer will disturb the equilibrium between the nematic andisotropic phases leading to different rates and magnitudes of nucleationat those sites. The differences between the nematic and isotropicregions are clearly detectable.

V. Patterned (ordered) Liquid Crystals

One approach to the patterning of the mesogenic layer on flat and curvedsurfaces is based on the use of patterned SAMs of molecules to directboth the polar (away from the surface) and azimuthal (in the plane ofthe surface) orientations of the mesogenic layer. This method is simpleand flexible, and any of the recently established procedures forpatterning SAMs on surfaces (for example, microcontact printing orphoto-patterning) (Talov et al., J. Am. Chem. Soc. 115: 5305 (1993);Kumar et al., Acc. Chem. Res. 28: 219 (1995), and references therein;Xia et al., J. Am. Chem. Soc. 117: 3274 (1995), and references thereincan be used; Jackman et al., Science 269: 664 (1995)). Using any ofthese methods, SAMs which pattern liquid crystals can be easily extendedto sizes ranging from hundreds of nanometers (Xia et al., J. Am. Chem.Soc. 117: 3274 (1995), and references therein) to millimeters and permitboth planar (parallel to the surface) and homeotropic (perpendicular tothe surface) orientation of mesogenic layers; methods based on therubbing of polymer films mainly provide manipulation of the in-planealignment of mesogenic layers and cannot homeotropically align mesogeniclayers. One class of useful SAMs has surface energies (˜19 mJ/m²) abouthalf those of films of polyimides used for alignment of liquid crystals;low-energy surfaces are less prone to contamination by molecularadsorbates and dust particles than are high-energy ones. Because SAMscan also be patterned on non-planar surfaces (Jackman et al., Science269: 664 (1995)), patterned mesogenic structures formed with SAMs can bereplicated on curved surfaces.

The capacity to pattern mesogenic layer orientations on nonplanarsurfaces provides procedures for the fabrication of tunable hybriddiffractive-refractive devices. For example, devices based oncombinations of diffractive and refractive optical processes permitaplanatic or chromatic correction in lenses, spectral dispersion,imaging from a single optical element, and other manipulations of light(Resler et al., Opt. Lett. 21, 689 (1996); S. M. Ebstein, ibid., p.1454; M. B. Stem, Microelectron. Eng. 32, 369 (1996): Goto et al., Jpn.J. Appl. Phys. 31, 1586 (1992); Magiera et al., Soc. Photo-Opt. Instrum.Eng., 2774, 204 (1996)). The capability to pattern mesogenic layers oncurved surfaces also provides routes for the fabrication of displayswith wide viewing angles.

In a presently preferred embodiment, the tunable hybrid device permitsthe manipulation of light. In a further preferred embodiment, the deviceis a refractive-diffractive device. In a still further preferredembodiment, the device permits imaging from a single optical element. Inyet another preferred embodiment, the device permits aplanatic orchromatic correction in lenses. In still another preferred embodiment,the device allows for spectral dispersion.

In another presently preferred embodiment, the SAM is layered on amaterial suitable for use as an electrode. In a preferred embodiment,the material is a metal film. In a further preferred embodiment, themetal film is a gold film.

The patterned mesogenic layers of the present invention can be tuned bythe use of electric fields. In a preferred embodiment, the electricfield is used to reversibly orient the mesogenic layer. In a stillfurther preferred embodiment, the electric field is applied eitherperpendicular to, or in the plane of, the surface of the mesogeniclayer. In another preferred embodiment, the oriented mesogenic layermodulates the intensity of light diffracted from the layer.

The discussion above, concerning SAM components, SAM components withreactive groups and SAM components bearing recognition moieties isequally applicable in the context of this aspect of the invention. Thus,the constituents of the SAM can be chosen from any of a wide variety ofappropriate molecules. In a presently preferred embodiment, the SAMcomprises mixtures of R²¹CH₂(CH₂)₁₄SH and R³¹CH₂(CH₂)₁₅SH, where R²¹ andR³¹ are independently members elected from the group consisting ofhydrogen, reactive groups and recognition groups, as discussed above.

VI. Analytical Devices

The device of the present invention can be of any configuration thatallows for the contact of a mesogenic layer with an organic layer orinorganic layer (e.g., metal, metal salt or metal oxide). The onlylimitations on size and shape are those that arise from the situation inwhich the device is used or the purpose for which it is intended. Thedevice can be planar or non-planar. Thus, it is within the scope of thepresent invention to use any number of polarizers, lenses, filters,lights, and the like to practice the present invention.

Although many changes in the mesogenic layer can be detected by visualobservation under ambient light, any means for detecting the change inthe mesogenic layer can be incorporated into, or used in conjunctionwith, the device. Thus, it is within the scope of the present inventionto use lights of various sources, microscopes, spectrometry, electricaltechniques and the like to aid in the detection of a change in themesogenic layer.

In those embodiments utilizing light in the visible region of thespectrum, the light can be used to simply illuminate details of themesogenic layer. Alternatively, the light can be passed through themesogenic layer and the amount of light transmitted, absorbed orreflected can be measured. The device can utilize a backlighting devicesuch as that described in U.S. Pat. No. 5,739,879, incorporated hereinby reference. Light in the ultraviolet and infrared regions is also ofuse in the present invention.

Thus, in another aspect, the invention provides a method for varying theoptical texture of a mesogenic layer comprising a haloorganosulfur. Theoptical texture of the mesogenic layer is controlled by selecting thehalogen content of the haloorganosulfur.

The following sections describe a number of novel devices according tothe present invention.

A. Coated Slides

In some embodiments, the assay devices of the present invention areproduced by coating the various assay formats herein on a substratethrough which light can pass (e.g., transparent and opaque substrates).The present invention is not limited to the use of any particular typeof substrate. Indeed, the use of a variety substrates is contemplated,including, but not limited to silica and quartz substrates. In someembodiments, the substrates have the same dimensions as commerciallyavailable slides (e.g., microscope slides), while in other embodiments,the dimensions of the substrates are varied to correspond to the end useor to the type of machine used to analyze the substrate (e.g., a platereader).

In preferred embodiments, areas of anisotropic order (i.e., analyticzones) are created on the slide prior to or after coating of the assayformat on the slide. In either case, the area of anisotropic orderorients liquid crystals. In preferred embodiments, the area ofanisotropic order occupies substantially all of the substrate surface,while in other embodiments, discreet areas of anisotropy are formed.Methods of creating such areas, which are described in more detailelsewhere herein, include, but are not limited to, oblique deposition ofmetals such as gold, rubbing the substrate surface with a soft materialsuch a cloth or an abrasive material, nano-abrasion with a gas streamcontaining abrasive particles (e.g., silica), etching with liquid, andimmobilization of proteins on the substrate following by rubbing.

The present invention is not limited to any particular number of areasof anisotropic order. Indeed, the number of such areas, or analyticzones, can vary from 1 to a plurality zones (e.g., from 1 to 1534zones). In some embodiments, the analytic zones correspond to thedimensions and spatial distribution of standard commercial multiwellplates. It is contemplated that this distribution of analytic zonesallows analysis with commercial multiwell plate readers. In otherembodiments, the analytic zones can be smaller or larger than thedimensions of wells in standard multiwell plates. In furtherembodiments, the analytic zones correspond to dimensions at least equalto the field of view of a microscope objective (e.g., a 5×, 10×, or 20×objective). It is contemplated that this configuration facilitatesintegration with automated microscopy systems.

In some preferred embodiments, substrates upon which areas of anisotropyhave been created are coated with a coating material. In someembodiments, the coating is accomplished through use of commerciallyknown procedures such as silk screen printing, flexographic printing,microcontact printing, seriagraphic printing, ink-jet printing, intaligoprinting, off-set printing, Heidelberg press printing, and thermal laserprinting. The present invention is not limited to the use of anyparticular coating material. Indeed, the use of a variety of coatingmaterials is contemplated, including hydrophobic coating materials,hydrophilic coating materials, coating materials containingelectroconductive elements, and coating materials containingelectroinsulating materials. Specific examples of such materialsinclude, but are not limited to, polyurethane, polyethylene, GORTEX(polytetrafluoroethylene), DACRON (polyethylene tetraphthalate), TEFLON(polytetrafluoroethylene), PVDF (polyvinylidene difluoride), proteinssuch as BSA, latex, polystyrene, silicone, cellulose, andnitrocellulose. In preferred embodiments, the coating material isapplied to form the assay formats described in more detail elsewhereherein. In still further embodiments, the coating material is applied soas to create microfluidic channels (see, e.g., U.S. Pat. No. 6,509,085,which is hereby incorporated by reference). Where necessary, the coatingmaterial is cured (e.g., by UV irradiation or infrared radiation)following application to the substrate.

The thickness of the coating material applied can vary. In somepreferred embodiments, the thickness is from about 1 micrometer to about100 micrometers, in more preferred embodiments, the thickness is fromabout 5 micrometer to about 35 micrometers, and in most preferredembodiments, the thickness is from about 20 micrometers to about 25micrometers.

In further preferred embodiments, a pigment is included in the coatingmaterial so that non-coated areas transmit light while coated areassubstantially block the transmission of light. The present invention isnot limited to the use of any particular pigment. Indeed, the use of avariety of pigments is contemplated, including black, red and bluepigments.

In some embodiments, a second untreated or treated substrate (e.g., aglass slide) is placed on the coated slide prior to curing. Pressure isthen applied to the substrates so that upon curing, the two substratesadhere to one another. In some embodiments, the pressure is applied bytransporting the paired substrates through a roller. The pairedsubstrates are then cured to produce an optical cell.

In still further embodiments, the coating material comprises a pressuresensitive adhesive. In some embodiments, the coating material comprisinga pressure sensitive adhesive defines one to a plurality of analyticzones. In some embodiments, the coating material comprising a pressuresensitive adhesive further comprises a pigment. In preferredembodiments, the pigment is black. In still further embodiments, thecoating material comprising a pressure sensitive adhesive comprises apolymeric coating material (e.g., acrylic or TEFLON polymer) to providea hybrid coating material. In some preferred embodiments, the pressuresensitive adhesive is low tack.

In other embodiments, the pressure sensitive adhesive is printed ontothe substrate after printing with another coating material. The patternof pressure sensitive adhesive printed onto the substrate can have avariety of configurations. For example, in some embodiments, thepressure sensitive adhesive is printed in a rectangular outline aroundan analytic zone. In other embodiments, the pressure sensitive adhesiveis applied in a plurality of points on the perimeter of the substrate.In still other embodiments, the pressure sensitive adhesive is appliedonly to the second untreated substrate that is used to cover the firstsubstrate comprising the analytic zone(s). In some embodiments, thepressure sensitive adhesive is a double-sided tape comprises a removablebacking that can be removed prior to assembly of the optical cell. Inother embodiments, the double sided tape does not comprise a removablebacking.

As described above, in preferred embodiments the coating material isapplied to create microfluidic channels. In some embodiments, themicrofluidic channels allow the fluidic movement of samples placed in anentry port on the substrate to an analytic zone. In some preferredembodiments, the top substrate comprises reservoirs for receivingsamples, rinse solutions, or liquid crystal solutions. The microfluidicchannels are in fluid communication with the reservoirs and terminate inoutlet ports. In preferred embodiments, the microfluidic channels are ofa dimension such that small variations in chamber pressure will notappreciably affect flow rates.

Motive force for delivery of fluids (e.g., samples, wash solution, orliquid crystals) via the microfluidic channels is supplied in a varietyof ways, including, but not limited to, syringe pumps, disposableMedicell pumps, placement of wicking materials at the outlet port, droppumping (i.e., movement from small drop to large drop), placement over afluid column over an inlet port, placement of positive atmosphericpressure at inlet ports and/or placement of negative atmosphericpressure at outlet ports. It is contemplated that positive pressure canbe delivered via use of a substantially sealed chamber in conjunctionwith an air pump or bottled gas (e.g., nitrogen gas). Likewise, negativepressure can be delivered via use of a substantially sealed chamber inconjunction with a vacuum pump. A vacuum may be applied by a variety ofmethods, including, but not limited to, use of running water and aventuri valve, AC or DC pumps, spring loaded negative syringe pump ordeformable sphere with elasticity such as a bulb syringe pipettor.

In some embodiments, assay devices comprising a substrate having atleast one microchannel therein are utilized to quantify the amount of ananalyte in a sample. It must be noted that these embodiments are notlimited to assay devices fabricated in any particular manner. Forexample, in some embodiments, the assay devices comprising microchannelsare fabricated by printing, while in other embodiments, the assaydevices comprising microchannels are fabricated by micromolding orlithography. In some embodiments, the microchannel comprises at leastone surface that orients a liquid crystal. Such surfaces may befabricated by a variety means, including, but not limited to,nanoabrasion, rubbing, and oblique deposition of metals. In furtherpreferred embodiments, the microchannel, including the surface thatorients a liquid crystal, is decorated with a recognition moiety.Methods for attaching recognition moieties are describes in detailabove. As needed, the substrate also preferably includes reservoirs andports for introducing samples, assay reagents (e.g., mesogens) and washsolutions into the microchannel. In each case, the reservoir or port isfluidically connected to the relevant microchannel.

In operation, a sample suspected of containing an analyte is allowed toflow through the microchannel under conditions where analytes present inthe sample can bind to the recognition moiety. Mesogens are then appliedto microchannel and the substrate is analyzed for the presence orabsence of liquid crystal ordering. Analysis of the liquid crystal canbe conducted by any of the means described in more detail herein,including, but not limited to, a microscope equipped with polarizinglenses and a plate reader. It is contemplated that areas where there isrecognition moiety-substrate binding will be identified by a lack oforder in the liquid crystal, while areas in which there is no bindingwill be identifiable by the presence of an ordered liquid crystal.Moreover, because the sample is allowed to flow along the length of themicrofluidic channel, binding is greater at the point entry into thechannel as opposed to the distal end of the channel. In other words, theamount of analyte in the sample is depleted as the sample flows alongthe channel. This effect may be used to quantify the amount of analytein the sample, which is proportional to the length of the microchannelover which liquid crystal order is disrupted.

In some embodiments, the substrate comprises a plurality ofmicrochannels so that serial dilutions of the sample may be applied inparallel to provide accurate quantitation of the amount of analyte in asample. In other embodiments, test samples are compared to controlsamples by running the samples in parallel in separate microfluidicchannels. The difference in areas of the microchannels over which liquidcrystal disorder is disrupted is proportional to the amount of analytein the test sample. Example 5 provides an example of this method.

B. Plate Readers

The present invention contemplates the use of plate readers to detectchanges in the orientation of mesogens upon binding of an analyte. Theplate readers may be used in conjunction with the LC assay devicesdescribed herein and also with the lyotropic LC assays described in U.S.Pat. No. 6,171,802, incorporated herein by reference. In particular, thepresent invention includes methods and processes for the quantificationof light transmission through films of liquid crystals based onquantification of transmitted or reflected light.

The present invention is not limited to any particular mechanism ofaction. Indeed, an understanding of the mechanism of action is notrequired to practice the present invention. Nevertheless, it iscontemplated that ordered nanostructured substrates impart order to thinfilms of liquid crystal placed onto their surface. These ordered filmsof liquid crystal preserve the plane of polarized light passed throughthem. If the liquid crystal possesses a well-defined distortion—such asa 90 degree twist distortion—then the liquid crystal will change thepolarization of the transmitted light in a well-defined and predictablemanner. It is further contemplated that ordered films of liquid crystaldifferentially absorb (relative to randomly ordered films of liquidcrystal) specific wavelengths of light.

In some embodiments of the present invention, the amount of targetmolecule or molecules bound to a sensing surface of an LC assay device(i.e., a surface decorated with a recognition moiety) increases with theconcentration/amount of target molecule present in a sample in contactwith a sensing surface. In preferred embodiments, the amount of boundtarget molecule changes the degree of disorder introduced into a thinfilm of liquid crystal that is ordered by nature of the underlyingnanostructured sensing substrate. In some embodiments, the degree oforder present in a thin film of liquid crystal determines the amount oflight transmitted through the film when viewed through crossed polars.In other embodiments, the degree of order present in a thin film ofliquid crystal determines the amount of light transmitted through thefilm when viewed using specific wavelengths of light. In still otherembodiments, the reflectivity of an interface to a liquid crystal canchange with the orientation of the liquid crystal. Therefore, in someembodiments, oblique illumination of the LC assay device is utilizedwith collection and analysis of reflected light being performed.

Accordingly, the present invention contemplates the use of plate readersto detect light transmission through an LC assay device when viewedthrough cross or parallel polars, the transmission of light through anLC assay device illuminated with a suitable wavelength of light, orreflection of light (i.e., polarized light or non-polarized light ofspecific wavelengths) from the surface of an LC assay device. Inparticularly preferred embodiments, plate readers are provided that aredesigned to be used in conjunction with LC assays. Other embodiments ofthe present invention provide modified commercially available readerssuch as ELISA readers and fluorometric readers adapted to read LCassays.

Non-limiting examples of the plate readers adapted for use in thepresent invention may be found in WO 03/019,191, which is hereinincorporated by reference. In preferred embodiments, two polarizingfilters are placed in the optical pathway of the plate reader in acrossed or parallel polar configuration. One filter is placed on theemission side of the light path prior to passing through the samplewhile a second polarizing filter is placed on the analyzing side of thelight path after light has passed through the sample but before it iscollected by a sensing devise such as a photodiode, a photomultiplier ora CCD. An ordered liquid crystal in the LC assay device preserves theplane of polarization and the amount of light reaching the lightgathering and sensing device is markedly attenuated when viewed throughcross polars or markedly accentuated when viewed through parallelpolars. Random organization of the liquid crystal of the LC assay devicedoes not preserve the plane of polarization and the amount of light,passing through crossed polars, reaching the light collecting andsensing device is relatively unaffected. Accordingly, in preferredembodiments, the binding of target molecules by the recognition moietiesin an LC assay device introduces disorder into the overlying thin filmof LC that increases with the amount of bound target molecule. In otherpreferred embodiments, the presence of a cell on an ordered regionintroduces disorder into the overlying LC. In other embodiments,specific bandpass filters are placed on the excitation side of the lightpath before light encounters the sample as well as on the emission sideof the light path (after light has passed through or is reflected by thesample but before reaching the light collecting and sensing device(e.g., photodiode, photomultipier or CCD). This configuration is usefulfor quantifying both reflected and transmitted light.

The present invention also provides LC assay devices configured for usein the plate reader. In preferred embodiments, the LC assay device isformatted or arrayed according to the dimensions of standardcommercially available plates (e.g., 24, 96, 384 and 1536 well plates).In some embodiments, the LC assay device comprises a surface (e.g., asubstrate with recognition moieties attached) that is of proper externaldimensions to be accurately fit into a given commercial reader. In someembodiments, the substrate contains uniform topography across itssurface, in other embodiments, the substrate contains a gradient oftopographies across its surface whereas in yet other embodiments regionsof topography are limited to discrete regions that correspond to areasread out by commercial plate readers. The recognition moieties may bearrayed on the substrate surface in any appropriate configuration. Forexample, in some embodiments, a single binding antibody, polypeptide orprotein, phospholipid, polynucleotide, or carbohydrate is evenlydistributed across the surface. In other embodiments, a single bindingantibody, polypeptide or protein, phospholipid, polynucleotide, orcarbohydrate is distributed across the surface in a gradient. In stillother embodiments, a single binding antibody, polypeptide or protein,phospholipid, polynucleotide, or carbohydrate is arrayed in discretespots that are in proper alignment to be read by the commercial reader.In still further embodiments, a variety of different single bindingantibodies, polypeptides or proteins, phospholipids, polynucleotides, orcarbohydrates are arrayed in spots that are in proper alignment to readby the commercial reader. In still other embodiments, a variety ofdifferent single binding antibodies, polypeptides or proteins,phospholipids, polynucleotides, or carbohydrates are arrayed in zonesalong the surface. Each zone contains a different polypeptide orprotein, cell receptor or binding sequence. The plate is read atpredetermined points (e.g., spots corresponding to the location of thewells in a 96 well plate). By designing the zones to the configurationof the plate reader it will be known which “well” readings correspond toeach zone. In other embodiments, specifically designed well inserts (tobe used with commercially available 6, 12, 24, 96, 384 or 1536 wellplates) containing the nanostructured sensing surface will be used inconjunction with commercially available multiwell plates for performingthe LC assays.

In some embodiments, plate readers are utilized to identify a reactionfront in a microfluidic channel. In these embodiments, a substrate isprovided in which the position of one or more microfluidic channels inthe substrate corresponds to the position of a row of wells in acommercial multiwell plate. The plate reader is then utilized to readdiscreet areas along the microfluidic channel. It is contemplated thatthis method will allow detection of how far along a microchannel that agiven reaction (i.e., binding of an analyte to a recognition moiety)occurs. The distance of the reaction can then be used to quantify theamount of analyte present. It will be recognized that the spatialresolution of the readout can be increased by reading more areas alongthe microfluidic channel. This can be accomplished by utilizing platereaders configured to read out 1534 well plates.

Additionally, some microplate readers have a scanning mode wherein thereading element performs a scan of assigned diagnostic regionscorresponding to well locations in a multiwell plate. The presentinvention contemplates the use of such readers to read out multiplelocations along a microfluidic channel so that a reaction front isidentified. Alternatively, the scanning reader is utilized to scan thelength of a microfluidic channel to identify a reaction front. Likewise,a plate reader can be used to measure a linear area of response on thesurface of a substrate exposed to an analyte. In this embodiment, thereaction is not confined to a microfluidic channel, but instead isallowed to proceed across one or more areas on a substrate surface.Multiple analytic zones programmed into the plate reader are then readout to determine the extent of the one or more areas over which areaction (e.g., disruption of liquid crystal order by migration ofcells) has occurred. Software can be modified to optimize both thelocation and movement of the sensing elements of the plate reader aswell as subsequent analysis of the biophotonic output. For example, thesensing elements are programmed to obtain multiple discreet readings orcontinuous linear scans from predetermined analytic zones currentlyemployed by commercial plate readers (e.g., corresponding to welllocations of 24, 96, 384 or 1536 plates) or could be programmed toobtain readings from novel locations that correspond to novel platedesigns for use with the assays described in detail in this document. Itwill be recognized that these devices and methods are not limited to usewith liquid crystal assays, but instead are broadly applicable todifferent assays such a fluorescent, calorimetric, and densitometricassays.

In some embodiments (for example, cell migration or movement assays),the plate reading device is configured to sample multiple regions withinin a given assay region. For example, the plate reader can be configuredto provide multiple circular readouts within a circular region definedby a well of a multiwell plate. Thus, the presence of cells can bedetected in regions that are remote from a central cell seeding area. Asanother example, the plate reader is configured to provides readouts inconcentric circles originating from a central cell seeding region. Inthis embodiment, the number of cells within each successive concentriccircle provides information as to the extent of migration (for example,in response to a test compound). The area under the curve for the signalfrom each successive concentric circle can be measured and plotted(signal vs. zone) to provide an analysis of strength of response to atest compound.

In other embodiments, the plate reading device is configuredasymmetrically sample a well or other assay region, for example, theright or left side of a central cell seeding zone. It is contemplatedthat such asymmetric sampling will yield data that distinguishedchemotaxis from chemokinesis. For example, if the number of cells in theright and left regions is equal, the compound is chemokinetic. If thecell signal is strongest in the region with the highest amount testcompound, then the compound is chemotactic. It will also be recognizedthat the plate reader can be configured as described above so that themultiple discrete regions are read within a given assay region.Chemokinesis is indicated by randomly distributed cells, whilechemotaxis is indicated by an increased number of cells in sample areasoriented closer to a test compound source as opposed to areas moreremote from a test compound source.

It will also be recognized that the present invention provides an assaysystem comprising a plate reading device and an LC assay device, whereinthe plate reading device and LC assay device are configured so thatlight provided from the plate reading device which is passed through orreflected from at least one surface of the LC assay device is detectedby a detection unit of the plate reading device. Suitable detectingunits include CCDs, photodiodes and photomultiplier tubes.

In other embodiments, imaging systems (e.g., array reading systems andgel readers) may be utilized that image the entire plate or a portionthereof (e.g., individual wells) at once. The data obtained from suchsystems is then processed to provide information on individual assayareas with the plates or wells. Such imaging systems can preferablyutilize optical imaging devices such as CCDs or other imaging devicessuch as magnetic resonance imagers.

Commercially available plate readers that may be modified according tothe present invention include, but are not limited, to those availablefrom Nalge Nunc International Corporation (Rochester, N.Y.), GreinerAmerica, Inc. (Lake Mary, Fla.), Akers Laboratories Inc., (Thorofare,N.J.), Alpha Diagnostic International, Inc. (San Antonio, Tex.), BiotekInstruments, Inc., (Winooski, Vt.), Tecan U.S. (Durham, N.C.), andQiagen Inc. (Valencia, Calif.).

3. Substrate Holder for Use with Multiwell Plate Readers

In some embodiments, the present invention provides substrate holdersfor use with multiwell plate readers. In preferred embodiments, thedimensions of the slide holder correspond to the dimensions ofcommercial multiwell plates so that the substrate (e.g., a slide oroptical cell made from slides) can be placed in the holder and theholder placed in a multiwell plate reader. In some embodiments, thesubstrate holder is configured to accept a plurality of substrates(e.g., 2-4 substrates). Once the substrate holder containing thesubstrate(s) is placed in plate reader, the analytical zones can be readby the plate reader. Accordingly, in preferred embodiments, theindividual substrates are held in such a position such that the analyticzones of the substrates correspond to the spatial location of wells incommercial multiwell plates. The substrate holder can be constructedfrom a variety of materials, including, but not limited to, stainlesssteel, titanium, aluminum, aluminum alloys, polymeric materials such asplexiglass or polycarbonate, wood, and glass.

One embodiment of an assay substrate holder (100) of the presentinvention is depicted in FIG. 48. The assay substrate holder (100)comprises a support surface (200). In preferred embodiments, thedimensions of the support surface (200) are substantially similar to thedimensions of a commercial multiwell plate. In other words, thefootprint of the support surface (200) is substantially similar to thefootprint of a commercial multiwell plate. In further preferredembodiments, the support surface (200) has at least one cutout (300)therein into which an assay substrate (400) can be inserted. In theembodiment shown, the assay substrate holder accommodates 4 assaysubstrates, one of which is shown inserted into the substrate holder.Preferably, the dimensions of the cutout are such that an open area(500) is provided along the side an inserted assay substrate (400) tofacilitate easy insertion and removal of the assay substrate from theassay substrate holder. The assay substrate (400) shown in FIG. 48 is aprinted liquid crystal assay substrate comprising a plurality of assayregions (600) that orient crystals which are defined by a coated surface(700)

It will be recognized that the substrate holder of the present inventionis not limited to use with the liquid crystal devices of the presentinvention. Indeed, the substrate holder finds use in a variety of assayformats, including, but not limited to, colorimetric, fluorimetric, andchemiluminescent assay formats. Thus, analytic zones of the substratecan be configured for use with each of these different assay formats.

4. Handheld Viewer for Use with Liquid Crystal Assay Devices

In some embodiments, the present invention provides a handheld viewerfor use in conjunction with the liquid crystal assay devices of thepresent invention. In preferred embodiments, the hand held viewercomprises an ocular loupe (e.g., an 8× or 10× loupe) with a holder thatallows placement of the assay device at the focal point of the loupe. Insome preferred embodiments, the slide holder further comprises a whitediffusing screen that allows for uniform illumination of analytic zoneson the substrate. In still further embodiments, the loupe comprises areticule that allows the user to measure the distance of a reaction(e.g., the distance a reaction occurs along a microfluidic channel). Infurther preferred embodiments, the holder allows movement of thesubstrate under the focal point of the loupe so that different analyticzones can be viewed.

VII. Cell Assays

The following sections further describe various embodiments of thepresent invention. The present invention is not intended to be limitedhowever to the following embodiments. Indeed, one skilled in the artwill be readily able to apply and adapt the disclosed embodimentsdirected to detecting cell migration, adhesion, proliferation, andcytological features for use in applications in other fields anddisciplines.

A. Cell Adhesion and Proliferation Assays

The present invention contemplates a number of embodiments useful foremploying liquid crystals for determining the number of cells present ona substrate. It is contemplated that these embodiments will allowperforming cell adhesion and cell proliferation assays. In preferredembodiments, the cell adhesion and cell proliferation assays areperformed on nanostructured substrates or substrates onto whichstructure is introduced by the seeding or decoration of the surface withnano- to micro-sized particles that order the LC layers applied thereto.

While not being limited to any particular mechanism or theory, thepresent invention contemplates that in these assays, the area occupiedby a cell is roughly equivalent to a planar surface as it would notorient a LC placed over its surface. Therefore, the number of cellspresent on a substrate will be proportional to the surface area coveredby the cells. It is contemplated that the exact relationship betweensurface area occupied by a given number of cells is dependent on thecell type and line used and the culture conditions employed.

In some preferred embodiments of the present invention, the areaoccupied by cells attached to an ordered substrate is characterized by anon-aligned (i.e., disordered) area of the liquid crystal. The areaoccupied by cells is thus quantifiable using a variety of methods. Inpreferred embodiments, the assay device is analyzed using cross polarsin conjunction with a CCD, photodiode or photomultiplier. With thissystem, the increased amount of light transmitted through the disorderedareas can be analyzed. In further preferred embodiments, specificwavelengths of light are used in conjunction with thin films of liquidcrystals to report the area occupied by cells.

In still other preferred embodiments of the invention, a liquidcrystalline substrate is prepared such that the presence of a cellattached to the surface of the liquid crystalline substrate leads to achange in the optical appearance of the substrate.

Substrates suitable for the cell adhesion, quantification, proliferationand migration assays include, but are not limited to, substrates havingrubbed protein surfaces, rubbed polymeric surfaces (e.g., tissue culturepolystyrene), ordered polymeric substrates formed by micromolding oflithographically created masters, oblique deposition of gold films, andnano- to micro-sized particles seeded onto the surface that are orderedupon initial deposition using a nanostamper or negative nanostamper orparticulate matter that is randomly seeded onto a surface andsubsequently ordered by motive forces, exemplified by, but not limitedto electric fields, magnetic fields and fluid flow. An additionalsubstrate suitable for cell adhesion and proliferation assays is aliquid crystalline substrate. The liquid crystalline substrate ispreferably prepared from a low molecular weight liquid crystal, apolymeric liquid crystal, a lyotropic or thermotropic liquid crystal, ora composite of liquid crystal and polymer, including biological polymerssuch as those that comprise the extracellular matrix.

In some particularly preferred embodiments, a biological moiety iscovalently or noncovalently associated with the surface of thesubstrate. Suitable biological moieties include, but are not limited to,sugars, proteins (e.g., extracellular matrix proteins such as collagen,laminins, fibronectin, vitronectin, osteopontin, thromospondin,Intercellular adhesion molecule-1 (ICAM-1), ICAM-2, proteoglycans suchas chondroitin sulfate, von Willebrand factor, entactin, fibrinogen,tenascin, Mucosal adressin cell adhesion molecule (MAdCAM-1), C3b, andMDC (metalloprotease/disintegrin/cysteine-rich) proteins), nucleicacids, specific receptors and cell receptor recognition sequences (e.g.,cadherein, immunoglobulin superfamily, selectin, mucin and integrinbinding sequences such as RGD, EILDV, LDV, LDVP, IDAP, PHSRN, SLDVP,GRGDAC, and IDSP)). In some embodiments, these biological moieties areassociated with a substrate or surface that is ordered. In otherembodiments, a surface or substrate with which biological moieties areassociated is ordered by a method such as rubbing. It is contemplatedthat using rubbed protein/peptide substrate surfaces in the celladhesion, migration, contraction and proliferation embodiments of thepresent invention allows researchers to investigate the impact of theseconstituents and to optimize assay conditions. For example, it iscontemplated that the use of rubbed protein substrates will promote theadhesion of seeded cells and also promote cell function (e.g., such asadhesion, contraction, proliferation and migration). However, in someembodiments, it may be desirable to study cell function independent ofthe interaction of the rubbed protein substrates, thus, some embodimentsemploy polymeric substrates. Still other embodiments of the presentinvention provide substrates that combine attached protein/peptidemoieties with non-biological forms of substrate functionalization andfabrication such as oblique deposition of gold and micromolded surfaces.

Some embodiments of the cell adhesion and proliferation assays of thepresent invention provide a plurality of distinct assay regions thatallow for replicates of experimental conditions and controls to be runsimultaneously. In still other preferred embodiments, the assay devicesof the present invention are designed to have a footprint that iscompatible with standard commercial plate readers (e.g., 24, 96, 384,1536 etc., well plates). In still some other embodiments, simplenanostructured inserts are provided for use with commercial plates andplate readers.

B. Cell Migration Assays

Certain embodiments of the present invention provide assays forqualitatively and/or quantitatively determining the migration (e.g.,random movement as well as attraction or repulsion) of cells on asubstrate under control conditions and in response to one or morecompounds of interest. In particular, the present inventioncontemplates, as described more fully below, a variety of assay formatsoptimized for distinguishing the positive, neutral or negativechemotactic and chemokinetic effects of one or more test compounds oncells of interest.

In some embodiments, the present invention provides cell invasionassays. It is contemplated that these assays are useful as an indicationof neoplastic transformation and relative aggressiveness (invasiveness)of a tumor type. These in vitro assays are used to establish theeffectiveness of therapeutic agents in preventing/minimizing invasion.Some preferred embodiments employ the use of nanostructured substratesin combination with extracellular matrices. FIG. 5 demonstrates a designwhereby invasion of the matrix (100) results in a loss of cells from theadjacent nanopatterned region (200) that would report the relativenumber of cells present. The decrease in cell number on the patternedsubstrate is indicative of successful invasion of the adjacent matrix.In some embodiments, cells are first seeded and then overlayed with asuitable matrix material (e.g., Matrigel or agar).

In some preferred embodiments, the extent of invasion of the ECM byplacement of the liquid crystals on the ECM is read out. The presentinvention is not limited to any particular mechanism of action. Indeed,an understanding of the mechanism of action is not necessary to practicethe present invention. Nevertheless, the process of invasion of thecells into the ECM leads to a change in the structure of the ECM that isreflected in the orientations of liquid crystals placed on to thesurface. In a still further preferred embodiment of the invention, theECM is prepared with a slightly anisotropic structure such that ituniformly orients the LC in the absence of invasion of the ECM by cells.Changes to the structure of the ECM caused by the invaded cells, lead toa disruption of the uniform orientation of the LC. In other embodimentsof the invention, the process of invasion of the cells into the ECMleads to the introduction of anisotropic structure that is reflected inan increase in the order of LC placed onto the surface.

In still other embodiments of the present invention, extracellularmatrices with nanopatterned surfaces that align liquid crystal films areprovided. Referring to FIG. 6, cells are seeded onto the right handsurface only (100). Invasion of the ECM (200) (that is rubbed, stamped,molded or decorated with aligned nano- to micro-sized particles on itssurface such that it aligns LC placed on its surface (300)) by cells(400) results in a change in light transmission through a LC film placedon its surface in the test areas (e.g., where cells are initially seededand in the ECM). In FIG. 6 it can be appreciated that invasion of theECM (200) will cause changes in the alignment of LC mesogens in both theinitial seeding region (100) and in the ECM (100). Changes in theinitial seeding region (100) occur due to a decrease in cells on itssurface thus providing a greater surface area for aligning LC mesogens.Changes in LC mesogen orientation in the ECM (200) occur due to subtlechanges in the matrix caused by cell invasion that will be translated tothe nanopatterned surface and subsequently in the mesogens contained inthe thin film of LC placed on its surface.

It is contemplated that this embodiment may also be employed in studiesof cell biomechanics where subtle changes in surface mechanics caused byprocesses exemplified by, but not limited to, cell adhesion, migrationand contraction are reported by alterations in LC orientation that areobservable by viewing with polarized light and the appropriate use offilters and by the use of certain wavelengths of light.

In another embodiment of the invention, hybrid three-dimensionalmatrices composed of ordered LC and of extracellular matrix (ECM)constituents are provided that would support cell function upon orwithin the matrix. In preferred embodiments, the hybrid matrix is formedby gelling an admixture of constituents (singly or together) exemplifiedby, but not limited to, mesogens, sugars, proteins (e.g., extracellularmatrix proteins such as collagen, laminin, fibronectin, vitronectin,osteopontin, thromospondin, Intercellular adhesion molecule-1 (ICAM-1),ICAM-2, proteoglycans such as chondroitin sulfate, von Willebrandfactor, entactin, fibrinogen, tenascin, Mucosal adressin cell adhesionmolecule (MAdCAM-1), C3b, and MDC(metalloprotease/disintegrin/cysteine-rich) proteins), nucleic acids,specific receptors or cell receptor recognition sequences (e.g.,cadherein, immunoglobulin superfamily, selectin, mucin and integrinbinding sequences such as RGD, EILDV, LDV, LDVP, IDAP, PHSRN, SLDVP,GRGDAC, and IDSP). In preferred embodiments, the gel process isconducted while applying an orienting electric field. This results in amatrix with aligned mesogens that are stable after gelling. It iscontemplated that this gelling procedure also orients the other matrixconstituents (depending on their relative charge and asymmetry of chargedistribution).)

The oriented hybrid composite can be prepared by using electric fields,magnetic fields, or by mechanical shearing of the composite. In someembodiments, commercially available basement membrane-like complexes(e.g., Matrigel™, which is harvested from a transformed cell line (EHS))are used as the ECM constituent admixed with the liquid crystallinespecies. The liquid crystals can be thermotropic or lyotropic liquidcrystals. If lyotropic liquid crystals, then preferred mesogens includenon-membrane disrupting surfactants, and discotic mesogens that are notmembrane disrupting.

In some preferred embodiments, cells are seeded adjacent to the matrixas illustrated in FIG. 6 or cells are seeded on the surface of thematrix and invasion of the matrix by the cells is indicated by a changein LC orientation.

In a preferred embodiment of the invention, a matrix is prepared suchthat it possesses an anisotropic structure. This anisotropic structureis induced by one of a number of methods that will be evident to thoseskilled in the art, including but not limited to mechanical shear, andthe use of electric and magnetic fields. The presence of the anisotropicstructure will lead to anisotropic optical properties of the matrix.These anisotropic optical properties can include birefringence anddichroism. The presence of invaded cells is detected by a change in theanisotropic optical properties of the matrix. These can be determined byusing polarized light and an analyzer or monochromatic and/or polarizedlight.

Referring again to FIG. 7, in some embodiments cells are seeded on theright initially. The loss of cells from this surface as they invade thematrix would be evident as well as their invasion causing a change in LCorientation in the hybrid (LC-ECM) matrix on the left. This hybridmatrix could also be used in conjunction with a planar (i.e.,non-orienting) or opaque substrate.

In the embodiment depicted in FIG. 7, the cells are seeded on thesubstrate on the right and a change in the hybrid matrix on the leftwill occur with invasion of cells that will manifest as a change inmesogen orientation embedded within the matrix. It can be seen that theregion in which cells are initially seeded is a nanostructured substratethat aligns mesogens in the LC film and will therefore report thepresence and relative number of cells. As the cells invade the hybridmatrix on the left a decrease in cell number on the right will bereported simultaneous with a change in the hybrid matrix caused by thecellular invasion. In some embodiments a chemotactic gradient will besupplied to the cells.

In other embodiments (depicted in FIG. 8) the cell seeding regioncomprises a substrate that is opaque to light or is simply notstructured as to cause alignment of liquid crystal mesogens. In thisembodiment, the presence of cells and their subsequent decrease innumber on the initial seeding region (100) will not be reported buttheir successful invasion of the hybrid matrix will.

In some embodiments (depicted in FIG. 9), cells (100) are seeded ontothe surface of the hybrid ECM-LC matrix (200) and invasion of the matrixoccurs from the surface. Invasion is detected as an alteration inmesogen orientation. The versatility of this unique hybrid matrix isevident in its ability to be used in assays investigating the dynamicsof cell adhesion, cell contraction and cell invasion. The presentinvention is not limited to any particular mechanism of action. Indeed,an understanding of the mechanism of action is not necessary to practicethe present invention. Nevertheless, it is contemplated that thealignment of the mesogens is disrupted by minute forces exerted upon(cells seeded on the surface) or into (cells invading the matrix) thematrix. In preferred embodiments, the change in mesogen alignment isreported by the alteration in the passage of polarized light or otherspecific wavelengths or combinations of wavelengths of light. In stillfurther preferred embodiments, the present invention providesmesogen-decorated matrices, preferably mesogen-decorated ECMs. Mesogensare attached to the matrix (e.g., ECM) by using the primary amines,carboxylic acids, and thiols that are available for reaction on thecomponents of the matrix (e.g., ECM).

In other embodiments, matrices incorporating mesogens are prepared byembedding a pre-aligned LC matrix (100) within a larger extracellularmatrix (ECM:200) that is allowed to gel (see, e.g., FIG. 10). Thepresent invention is not limited to any particular mechanism of action.Indeed, an understanding of the mechanism of action is not necessary topractice the present invention. Nevertheless, it is contemplated thatchanges in the ECM are translated to the LC matrix. In otherembodiments, the ECM is dissolved into the mesogens when they are intheir isotropic state, and then the mesogens are transformed into theliquid crystalline state. The transformation of state can be achieved bychange of temperature and/or the evaporation of a solvent. Thisprocedure leads to the formation of a liquid crystal/ECM composite,which has the properties of a liquid crystal gel. Liquid crystal gelsare known by those skilled in the art to be prepared by using lowmolecular weight and high molecular weight gelators. In some preferredembodiments, the ECM is the commercially available MATRIGEL (See, e.g.,Monvoisin A. Bisson C. Si-Tayeb K. Balabaud C. Desmouliere A. RosenbaumJ.; Involvement of matrix metalloproteinase type-3 in hepatocyte growthfactor-induced invasion of human hepatocellular carcinoma cells;International Journal of Cancer 97(2):157-62, 2002).

In other preferred embodiments, the hybrid mesogen/matrices comprisetype I collagen and/or porcine submucosal matrix (See, e.g., Rosenthalet al., The mucosal invasion model: a novel in vitro model forevaluating the invasive behavior of mucocutaneous malignancies. Archivesof Otolaryngology—Head & Neck Surgery. 127(12):1467-70, 2001; Galvez etal., Membrane type 1-matrix metalloproteinase is activated duringmigration of human endothelial cells and modulates endothelial motilityand matrix remodeling. Journal of Biological Chemistry.276(40):37491-500, 2001.) While these matrices are the most widely usedmatrices for invasion assays, the present invention is not limited totheir use. Indeed, the use of other matrices is contemplated, including,but not limited to, Amgel-derived from human amniotic membranes (See,e.g., Siegal et al. Development of a novel human extracellular matrixfor quantitation of invasiveness of human cells. Cancer letters 69:123-32. 1993).

It is contemplated that the hybrid LC-matrices have numerous other usesin addition to invasion assays. The LC-matrices find use in studies ofcell biomechanics where subtle changes in the matrix caused by processes(e.g., cell adhesion, cell migration and cell contraction) are reportedby alterations in LC orientation. In preferred embodiments, suchalterations are detected by viewing the LC-matrix with filteredpolarized light. Additionally, since the orientation of LC is verysensitive to temperature shifts, the LC-matrices are useful forreporting changes in metabolic activity of individual and populations ofcells. In preferred embodiments, liquid crystalline substrates can alsobe used to report stresses imparted to substrates by cells adhered tosurfaces. In this embodiment of the invention, the cells are supportedon the surface of the liquid crystalline substrate. The substrates canbe decorated with ECM.

Similarly, an ECM that has a patterned surface (FIG. 6) can be used toreport changes induced in the surface (adjacent to the surface occupiedby cells) caused by events exemplified by but not limited to adhesion,migration and contraction. These changes are then reported by a LC filmplaced on its surface and subsequently viewed with polarized light orspecific wavelengths of light.

In still further embodiments, the substrates and hybrid matrices of thepresent also find use in contraction assays. The most common form ofcontraction assays for which the substrates and hybrid matrices areuseful are gel contraction assays (See, e.g., Fireman et al.,Morphological and biochemical properties of alveolar fibroblasts ininterstitial lung diseases. Lung. 179(2):105-17, 2001; Ballas et al.,Delayed wound healing in aged rats is associated with increased collagengel remodeling and contraction by skin fibroblasts, not with differencesin apoptotic or myofibroblast cell populations. Wound RepairRegeneration. 9(3):223-37, 2001; Roy et al., Exertion of tractionalforce requires the coordinated up-regulation of cell contractility andadhesion. Cell Motility & the Cytoskeleton. 43(1):23-34, 1999; and Leeet al., Extracellular matrix and pulmonary hypertension: control ofvascular smooth muscle cell contractility. American Journal ofPhysiology. 274(1 Pt 2):H76-82, 1998.

Single cell contraction is also sometimes evaluated by measuring crosssectional area after contraction is maximally stimulated in vitro. (See,e.g., Pang et al., Single-cell contraction assay for human ciliarymuscle cells. Effect of carbachol. Investigative Opthalmology & VisualScience. 34(5):1876-9, 1993. Another assay describes the forces exertedby individual cells calculated from time-lapse videomicroscopicrecordings of the 2-D elastic distortion of the matrix. See, e.g., Royet al. An in vitro force measurement assay to study the early mechanicalinteraction between corneal fibroblasts and collagen matrix.Experimental Cell Research. 232(1):106-17, 1997.

The present invention also provides methods and compositions forreporting biomechanical transduction events in cells of interest. Forexample, in certain embodiments, suitable liquid crystal membranes areused to nonspecifically report biomechanical transduction eventsassociated with cell adhesion, migration and contraction. While notbeing limited to any particular mechanism or theory, the presentinvention contemplates that in these embodiments, the detectable eventis the change in the order of the crystalline membrane itself. A numberof non-limiting embodiments of contemplated cell transduction assays arepresented in the following examples. In one example, a film of liquidcrystal is associated with a phospholipid which is subsequentlyspontaneously adsorbed by the cell of interest. In another example, anelastomeric liquid crystalline material is used and contacted to thecells of interest. In yet another example, a polymer-stabilized orpolymer-dispersed liquid crystal is used and contacted to the cells ofinterest. In another example, a liquid crystalline gel is used andcontacted with the cells of interest. In still another example, acollagen gel that has been micromolded such that it aligns liquidcrystals is used and contacted to the cells of interest. In thisembodiment, the cells of interest are allowed to attach to a collagengel and then incubated for a period of time which is dictated byexperimental design, cell type and culture conditions. Changes in stressimparted by cell contraction create detectable changes in the liquidcrystal layers. Yet other embodiments described below utilize a novelhybrid extracellular matrix-liquid crystalline composite for thereporting of biomechanical transduction events.

FIG. 11 demonstrates an embodiment of the invention that allows theevaluation of contractile forces by large populations of cells as wellas by individual cells. Cells (300) are seeded on a nanostructuredsurface (100) of extracellular matrix (200) that aligns liquid crystalmesogens placed on its surface. The contraction of a cell (400) resultsin altering the regular array of the nanostructured surface (500) whichin turn is reported by alterations in the alignment of LC mesogens.Cells may be imaged simultaneously as interpretation of LC alignmentchanges by staining the cells with vital or non-vital dyes (fluorometricand non-fluorometric). Dual exposure (with and without polarized light)allows localization of individual cells and correlating their locationand appearance with observed changes in LC alignment.

Additionally, the use of these matrix-LC interactions and hybridmatrices allows for examining the forces of individual cells seeded ontotheir surfaces, which provides insight into the dynamics of celladhesion, migration and contraction.

The present invention contemplates that cells migrating across asubstrate produce nanomechanical forces that either disorder an orderedsurface or order a disordered surface. In both cases, the change insurface order created by the passage of cells is detected and reportedby liquid crystal films. Migrating cells also sweep ordered surfaceparticles out of their path during migrations and introduce disorder inthe LC film placed on top subsequent to migration. Another variation isthe use of a nanostructured substrate with randomly distributed (seeded)and randomly oriented (relative to the long axis) nano- to micro-sizedparticles across its surface. This creates closely spaced regions thatintroduce disorder into the LC film effectively partially masking theordering influence of the underlying nanostructured substrate. Theseeding densities are such that many particles are present on thesurface relative to the size of a single cell. The migration of cellsunmasks the underlying nanostructured substrate and creates tracks ofpurely ordered mesogens. The particles can be any nano- to micro-sizedinert material including but not limited to colloidal gold, othermetals, pumice, graphite, silica, and glass beads. It is contemplatedthat the particles decorating the surface can be made more resistant todisplacement during the flow of LC over the surface (i.e., to preventthe particles from being washed off) by the adjunctive use ofstabilizing forces exemplified by, but not limited to, the use ofexternally applied electric fields (with charged particles), magneticfields (with magnetic particles), or chemical and physical treatment ofthe surfaces of the particles to make them adherent to the substrates

The assay devices of the present invention are designed to detect thesechanges in order or disorder. Accordingly, in some embodiments, asubstrate is provided that has at least one assay region that eitherorients or does not orient a liquid crystal. In some preferredembodiments, the assay region is associated with one or more biologicalmoieties as described in more detail below. However, the assay regionsneed not be necessarily associated with a biological moiety. In furtherpreferred embodiments, the assay regions are arranged in an array. Insome particularly preferred embodiments, the array is aligned withreadout zones of a plate reading device. In some embodiments of thepresent invention, the assay devices are further provided with a numberof topological features. For example, in some embodiments, an assayregion of the device has a depression or well formed in the substratefor containing cells, media, or test compounds.

In other preferred embodiments the assay devices further comprise a testcompound region or plurality of test compound regions separate from theassay regions. These regions are useful for providing one or more testcompounds for use in the assays and causing delivery of the one or moretest compounds to the assay regions. In some embodiments, the testcompound regions are peripherally located on the assay device ascompared to the assay regions, while in other embodiments, the testcompound regions are centrally located as compared to the assay regions,while in still further embodiments, the test compound regions areinterspersed with the assay regions. Various combinations of theselayouts are also contemplated by the present invention.

In some embodiments, the test compound regions are arranged to provide agradient (e.g., a concentration gradient) of at least one test compoundto the assay region. The gradients for two or more test compounds can bein the same direction or in different directions. In other embodiments,the test compound regions are arranged to deliver a relatively constantamount of at least one test compound to assay region(s). In someparticularly preferred embodiments, the test compound regions aredesigned to provide an analysis of whether a cellular response to aparticular test compound placed in a reservoir is chemotactic orchemokinetic in nature. In some preferred embodiments, the test compoundregions are channels, microchannels, or wells formed in the substrate.

In other preferred embodiments, the test compound regions are in theform a porous or nonporous material that releases a given test compoundinto the assay device. Suitable test compounds but are not limited to,small organic compounds, amino acids, vitamins and peptides andpolypeptides, including, but not limited to, magainin (e.g., magainin I,magainin II, xenopsin, xenopsin precursor fragment, caerulein precursorfragment), magainin I and II analogs (PGLa, magainin A, magainin G,pexiganin, Z-12, pexigainin acetate, D35, MSI-78A, MG0 [K10E, K11E,F12W-magainin 2], MG2+[K10E, F12W-magainin-2], MG4+[F12W-magainin 2],MG6+[f12W, E19Q-magainin 2 amide], MSI-238, reversed magainin II analogs[e.g., 53D, 87-ISM, and A87-ISM], Ala-magainin II amide, magainin IIamide), cecropin P1, cecropin A, cecropin B, indolicidin, nisin,ranalexin, lactoferricin B, poly-L-lysine, cecropin A (1-8)-magainin II(1-12), cecropin A (1-8)-melittin (1-12), CA(1-13)-MA(1-13),CA(1-13)-ME(1-13), gramicidin, gramicidin A, gramicidin D, gramicidin S,alamethicin, protegrin, histatin, dermaseptin, lentivirus amphipathicpeptide or analog, parasin I, lycotoxin I or II, globomycin, gramicidinS, surfactin, ralinomycin, valinomycin, polymyxin B, PM2 [(+/−)1-(4-aminobutyl)-6-benzylindane], PM2c[(+/−)-6-benzyl-1-(3-carboxypropyl)indane], PM3[(+/−)1-benzyl-6-(4-aminobutyl)indane], tachyplesin, buforin I or II,misgurin, melittin, PR-39, PR-26, 9-phenylnonylamine, (KLAKKLA)n,(KLAKLAK)n, where n=1, 2, or 3, (KALKALK)3, KLGKKLG)n, and KAAKKAA)n,wherein N=1, 2, or 3, paradaxin, Bac 5, Bac 7, ceratoxin, mdelin 1 and5, bombin-like peptides, PGQ, cathelicidin, HD-5, Oabac5alpha, ChBac5,SMAP-29, Bac7.5, lactoferrin, granulysin, thionin, hevein andknottin-like peptides, MPG1, 1bAMP, snakin, lipid transfer proteins,Insulin, Insulin like Growth Factors such as IGF-I, IGF-II, and IGF-BP;Epidermal Growth Factors such as α-EGF and β-EGF; EGF-like moleculessuch as Keratinocyte-derived growth factor (which is identical to KAF,KDGF, and amphiregulin) and vaccinia virus growth factor (VVGF);Fibroblast Growth Factors such as FGF-1 (Basic FGF Protein), FGF-2(Acidic FGF Protein), FGF-3 (Int-2), FGF-4 (Hst-1), FGF-5, FGF-6, andFGF-7 (identical to KGF); FGF-Related Growth Factors such as EndothelialCell Growth Factors (e.g., ECGF-α and ECGF-β); FGF- and ECGF-RelatedGrowth Factors such as Endothelial cell stimulating angiogenesis factorand Tumor angiogenesis factor, Retina-Derived Growth Factor (RDGF),Vascular endothelium growth factor (VEGF), Brain-Derived Growth Factor(BDGF A- and -B), Astroglial Growth Factors (AGF 1 and 2),Omentum-derived factor (ODF), Fibroblast-Stimulating factor (FSF), andEmbryonal Carcinoma-Derived Growth Factor; Neurotrophic Growth Factorssuch as α-NGF, β-NGF, γ-NGF, Brain-Derived Neurotrophic Factor (BDNF),Neurotrophin-3, Neurotrophin-4, and Ciliary Nuerotrophic Factor (CNTF);Glial Growth Factors such as GGF-I, GGF-II, GGF-III, Glia MaturationFactor (GMF), and Glial-Derived Nuerotrophic Factor (GDNF);Organ-Specific Growth Factors such as Liver Growth Factors (e.g.,Hepatopoietin A, Hepatopoietin B, and Hepatocyte Growth Factors (HCGF orHGF), Prostate Growth Factors (e.g., Prostate-Derived Growth Factors[PGF] and Bone Marrow-Derived Prostate Growth Factor), Mammary GrowthFactors (e.g., Mammary-Derived Growth Factor 1 [MDGF-1] and MammaryTumor-Derived Factor [MTGF]), and Heart Growth Factors (e.g.,Nonmyocyte-Derived Growth Factor [NMDGF]); Cell-Specific Growth Factorssuch as Melanocyte Growth Factors (e.g., Melanocyte-Stimulating Hormone[α-, β-, and γ-MSH] and Melanoma Growth-Stimulating Activity [MGSA]),Angiogenic Factors (e.g., Angiogenin, Angiotropin, Platelet-DerivedECGF, VEGF, and Pleiotrophin), Transforming Growth Factors (e.g., TGF-α,TGF-β, and TGF-like Growth Factors such as TGF-β₂, TGF-β₃, TGF-e, GDF-1,CDGF and Tumor-Derived TGF-β-like Factors), ND-TGF, and Human epithelialtransforming factor [h-TGFe]); Regulatory Peptides with GrowthFactor-like Properties such as Bombesin and Bombesin-like peptides(e.g., Ranatensin, and Litorin], Angiotensin, Endothelin, AtrialNatriuretic Factor, Vasoactive Intestinal Peptide, and Bradykinin;Cytokines such as the interleukins IL-1 (e.g., Osteoclast-activatingfactor [OAF], Lymphocyte-activating factor [LAF], Hepatocyte-stimulatingfactor [HSF], Fibroblast-activating factor [FAF], B-cell-activatingfactor [BAF], Tumor inhibitory factor 2 [TIF-2], Keratinocyte-derivedT-cell growth factor [KD-TCGF]), IL-2 (T-cell growth factor [TCGF],T-cell mitogenic factor [TCMF]), IL-3 (e.g., Hematopoietin,Multipotential colony-stimulating factor [multi-CSF], Multilineagecolony-stimulating activity [multi-CSA], Mast cell growth factor [MCGF],Erythroid burst-promoting activity [BPA-E], IL-4 (e.g., B-cell growthfactor I [BCGF-I], B-cell stimulatory factor 1 [BSF-1]), IL-5 (e.g.,B-cell growth factor II [BCGF-II], Eosinophil colony-stimulating factor[Eo-CSF], Immunoglobulin A-enhancing factor [IgA-EF], T-cell replacingfactor [TCRF]), IL-6 (B-cell stimulatory factor 2 [BSF-2], B-cellhybridoma growth factor [BCHGF], Interferon β₂ [IFN-B], T-cellactivating factor [TAF], IL-7 (e.g., Lymphopoietin 1 [LP-1], Pre-B-cellgrowth factor [pre-BCGF]), 1L-8 (Monocyte-derived neutrophil chemotacticfactor [MDNCF], Granulocyte chemotatic factor [GCF],Neutrophil-activating peptide 1 [NAP-1], Leukocyte adhesion inhibitor[LAI], T-lymphocyte chemotactic factor [TLCF]), IL-9 (e.g., T-cellgrowth factor III [TCGF-III], Factor P40, MegaKaryoblast growth factor(MKBGF), Mast cell growth enhancing activity [MEA or MCGEA]), IL-10(e.g., Cytokine synthesis inhibitory factor [CSIF]), IL-11 (e.g.,Stromal cell-derived cytokine [SCDC]), IL-12 (e.g., Natural killer cellstimulating factor [NKCSF or NKSF], Cytotoxic lymphocyte maturationfactor [CLMF]), TNF-α (Cachectin), TNF-β (Lymphotoxin), LIF(Differentiation-inducing factor [DIF], Differentiation-inducingactivity [DIA], D factor, Human interleukin for DA cells [HILDA],Hepatocyte stimulating factor III [HSF-111], Cholinergic neuronaldifferentiation factor [CNDF], CSF-1 (Macrophage colony-stimulatingfactor [M-CSF]), CSF-2 (Granulocyte-macrophage colony-stimulating factor[GM-CSF]), CSF-3 (Granulocyte colony-stimulating factor [G-CSF]), anderythropoietin; Platelet-derived growth factors (e.g., PDGF-A, PDGF-B,PDGF-AB, p28-sis, and p26-cis), and Bone Morphogenetic protein (BMP),neuropeptides (e.g., Substance P, calcitonin gene-regulated peptide, andneuropeptide Y), and neurotransmitters (e.g., norepinephrine andacetylcholine).

Accordingly, in some embodiments, the present invention provides anassay apparatus comprising a surface having at least one discrete assayregion thereon and wherein the assay region is associated with at leastone test compound formulated for controlled release. In someembodiments, the test compound formulated for controlled release isprovided in a matrix. In some embodiments, the matrix is a polymer.Various polymers that find use for controlled release applications,include, but are not limited to chitosan, chitosan-alginate,poly(N-isopropylacrylamide) hydrogels, lipid microspheres, copolymers ofpolylactic and polyglycolic acid, dextran hydrogels, and poly(ethyleneglycol) hydrogels. (See, e.g., Zambito et al., Acta Technol. Et LegisMedicamenti 14(1):1-11 (2003); Bhopaktar et al., Advances Chitin Sci.5:166-170 (2002); Zhuo et al., J. Polymer Sci. 41(1):152-159 (2002); DelCurto et al., Proceedings of the 28th Symposioum on Controlled Releaseof Bioactive Materials, San Diego, Calif., 2:976-977 (2001); Hu et al.,J. Drug Targeting 9(6):431-438 (2001); Lambert et al., J. ControlledRelease 33(1):189-195 (1995); Hennink et al., J. Controlled Release48(2,3):107-114 (1997); and Zhoa et al., J. Pharm. Sci. 87(11):1450-1458(1998). In some embodiments, the matrix further comprises anextracellular matrix component (e.g., collagen, vitronectin,fibrionectin or laminin. A variety of test compounds may be provided inthe matrix, including, but not limited to, polypeptides, carbohydrates,amino acids, and small organic compounds. These assay devices may beused with any of the read out and labeling methods described herein,including LC based assays, calorimetric assays, fluorimetric assays,opical density assays, and light scattering assays. In otherembodiments, the assay devices are configured with a plurality assayregions corresponding spatially to the wells of 6, 12, 24, 36, 96, 384or 1536 well plates. The matrix containing the test compound may beprovided in a variety of orientations, for example on the bottom of awell or other assay region, on the side of a well, as a strip in thebottom or side of well or other assay region, or as a bead on aninterior ssurface of a well or on an assay region.

In still further embodiments, the present invention provides kitscomprising an assay apparatus comprising a surface having thereon atleast one discreet asay region and unpolymerized matrix material. Insome embodiments, the discreet assay region further comprises a cellseeding region. In some embodiments, the kits provide instructions forpolymerizing the matrix material in the presence of at least one testcompound, applying the matrix to an apparatus, and culturing cells inthe apparatus. It is contemplated that foregoing apparatuses find use inassaying the response of cells to a stimulus from a test compound. Theapparatuses may also be utilized in high-throughput settings to measurethe effect of a panel or library of compounds on cells.

In still further embodiments, test compound regions are provided by thedifferential movement of materials (e.g., test compounds) by themanipulation of electrical fields, thermal gradients, and capillaryaction on the substrate surface. In other preferred embodiments of theinvention, chemotactic or chemokinetic agents are immobilized on thesurfaces. These agents can be presented in uniform concentration on asurface, they can be patterned on a surface or they can be present in agradient in concentration across a surface. The agents immobilized onthe surface may be released from the surface to make them available tothe cells by using changes in the microenvironment of the surfacescaused by the cells to trigger the release of the agents, or externallycontrolled variables (such as illumination or applied electricalpotentials) can be used to regulate the release of the agents from thesurface. In other preferred embodiments of the invention, the agents arenot released from the surface but interact with constituents of themembrane of the cell and thereby influence cell behavior.

In some further preferred embodiments, the assay regions of the devicesare associated with a biological moiety. In some embodiments, adisordered (e.g., randomly ordered) substrate or assay region on asubstrate appropriate for assays disclosed herein is created byattaching (e.g., covalently or noncovalently) one or more biologicmoieties, (e.g., sugars, proteins (e.g., extracellular matrix proteinssuch as collagen, laminin, fibronectin, vitronectin, osteopontin,thromospondin, Intercellular adhesion molecule-1 (ICAM-1), ICAM-2,proteoglycans such as chondroitin sulfate, von Willebrand factor,entactin, fibrinogen, tenascin, Mucosal adressin cell adhesion molecule(MAdCAM-1), C3b, and MDC (metalloprotease/disintegrin/cysteine-rich)proteins), nucleic acids, specific receptors or cell receptorrecognition sequences (e.g., cadherein, immunoglobulin superfamily,selectin, mucin and integrin binding sequences such as RGD, EILDV, LDV,LDVP, IDAP, PHSRN, SLDVP, GRGDAC, and IDSP)) onto a suitable substratesurface. In another embodiment, an ordered substrate or assay region ona substrate is created by covalently or noncovalently binding one ormore or the previously described biological moieties to a polymericsurface and subsequently rubbing the surface to create order. Thepresent invention is not intended to be limited by the order of stepstaken in creating a suitable substrate surface. For example, in someembodiments, the substrate is ordered prior to the attachment ofbiological moieties. In other embodiments, the substrate is orderedafter addition of biological moieties. Indeed, a number of processingevents and steps are adaptable to producing suitable substratecompositions for use in the assays disclosed herein given the specificguidance provided and the skill of those in the art.

In other embodiments, an ordered substrate is created by contacting asuitable surface with a plurality of evenly distributed particles (e.g.,magnetic nanoparticles) that when aligned orient a mesogenic layer. Asdescribed in detail above, the particles may be applied to the surface(positive nanostamp) or removed from the surface (negative nanostamp)with nanostamping devices (ref. FIGS. 1A and 1B). In particularlypreferred embodiments, the particles are magnetic nanoparticles that arealigned using a magnetic field. In another preferred embodiment, themetallic nanorods are small enough to be readily displaced by migratingcells.

In some embodiments of the present invention, the extent of overall cellmovement in the assay device is determined by analyzing the proportionof the substrate on which surface order is altered (e.g., a change fromordered to disordered and vice versa). Preferred methods for analyzingthe extent of areas of order and disorder are described in detail aboveand include the use of systems comprising two polarizers, CCDs,photomultipliers, photodiodes, plate reading devices, and analysis ofthe transmission of certain wavelengths of light. Other methods foranalyzing the results of the assays are described below.

A number of particularly preferred assay devices and systems will now bedescribed. It will be understood that the present invention is notlimited to these particular embodiments. One embodiment of the presentinvention to distinguish chemotactic/chemokinetic effects is illustratedin the model assay depicted in FIG. 13. Element (100) represents asuitable substrate (either ordered or disordered). A test compoundreservoir (200) is provided on the left edge of substrate (100). In someembodiments, reservoir (200) and substrate (100) are engineered so thatthe test compound is slowly transported or diffused across the substrate(100) and the assay region to contact cells (300) on the surface of thesubstrate. It is contemplated that convection can be minimized by usingvery thin films of liquid, by making the culture medium viscous throughthe addition of polymers, or by placing the whole system into a gel thatquenches convection but permits movement of the cells. In otherembodiments, these elements are engineered so that the test compound ismore quickly dispersed into the media covering cells (300) via capillaryaction. In performance of the assay, cells of interest (300) are seededonto the assay region on substrate (100). In one embodiment, the effectsof a test compound on cell motility can be determined as follows. Thetest compound is placed in reservoir (200) and cells (300) are againseeded in the assay region or randomly distributed across the entiresubstrate.

In some embodiments, a specifically designed reservoir is utilized toprovide a gradient of test compound(s) delivered through diffusion. FIG.14 demonstrates one possible design whereby a test compound(s) is placedinto a reservoir (100) and is allowed to diffuse along a thin tube (200)and exit from a small open port (300) in close proximity to the testsubstrate. The actual dimensions of the device are variable depending onthe needs of the specific assay and the test surface area (andaccompanying volume of media covering it) to which test compound isbeing delivered. The composition of the reservoir and the delivery tubecan be of a variety of materials including but not limited to glass,polypropylene, polystyrene, and silicone.

The reservoir device may be incorporated into the intrinsic design of anassay device or may be provided as an insert for use with commerciallyavailable or custom designed multiwell plates. It will be recognizedthat this assay design is not limited to use with liquid crystal assays,but also finds use with calorimetric, fluorometric, densitometric, andother assay formats.

If the test compound is chemotactic, the cell pathways are substantiallylinear with the net migration being towards the reservoir port (300 inFIG. 14). Movement of cells within the assay region 300 creates areas ofdisorder (on an ordered substrate) or introduces order by mechanicalmovement of cells across the surface of a randomly ordered substratethat can be assayed as described above. In particularly preferredembodiments, image analysis software is used to determine the relativelinearity of migration paths.

Is it also possible to formulate these test compounds into a “slowrelease tablet” that is placed at some location of the test surface. Thegradual dissolution of the tablet generates a locally high concentrationof the text compound. Some slow release tablets are placed underexternal control; for example; rates of dissolution are controlled byillumination of electrical potentials.

It will also be recognized that the device does not necessarily need tocomprise the reservoir (200). As a non-limiting example of an assay notrequiring the reservoir (200) the substrate (100) is prepared by seedingcells (300) in the assay region and providing the test compound in theaccompanying cell media. A compound that increases cellular activitywill have a positive effect on cell motility as compared to controlcompounds. In preferred embodiments, the assay region is ordered toorient liquid crystals. Movement of cells (300) within the assay regioncreates areas of disorder that can be assayed as described above. Insome embodiments, the analysis of separate assays involving addition ofa test compound to the test compound region or generally into the mediacan be used to differentiate the chemotactic and/or chemokinetic effectsof the test compound.

FIG. 15 shows yet another assay system for studying the effects of atest compound on cell motility and migration. The substrate (100)comprises a first region 200 and a second region 300 having distinctsurface order. In some embodiments, the first region (200) is ordered toorient liquid crystals while the second region (300) does not orientliquid crystals. In a preferred embodiment, substrate (100) alsocomprises a test compound reservoir (400).

One contemplated protocol using the assay system shown in FIG. 15 is asfollows. The user seeds cells in a uniform fashion onto the first andsecond regions (200 and 300). A test compound is introduced to substrate(100) in the media accompanying the seeded cells, provided via the testcompound region (400). Quantification of cell migration can be performedby measuring the number of cells present in the first region (200) bymeasuring the number of cells in the second region (300) or by comparingthe cell numbers present in the first region (200) and second region(300). If the test compound is chemokinetic, there should not be amarked change (increase or decrease) in the number of cells in the firstregion (200) or in the second region (300). However, if the testcompound is chemotactic there will be a marked increase in the number ofcells in the first region (200) and a decrease in the cells present inthe second region (300). If a compound has a negative effect onchemotaxis then there will be an increase in cell number in the secondregion (300) with a concomitant decrease in cell number in the firstregion (200).

In still another embodiment, as shown in FIG. 16, a substrate (100) isprovided having a reservoir (200) and two separate regions (300 and 400)of distinct surface order. Accordingly, in certain embodiments,substrate (100) further provides a first region (300) of orderedsubstrate that aligns liquid crystals, and a second region (400) ofsubstrate that does not align liquid crystals. In further embodiments, acell seeding region (500) (exemplified by but not limited to adepression or well) is provided for seeding cells of interest onsubstrate (100). In some embodiments of the present invention, methodsare provided wherein cells are seeded at the cell seeding region (500)on two separate substrates. Each group of seeded cells is allowed toattach and grow for a sufficient period of time (determined by cell typeand culture conditions) usually from 1-24 hr. The media is then replacedon each substrate, and sufficient fresh media is added to cover eachsubstrate. The test compound is then placed either in the media coveringthe substrate in a first instance, or in the reservoir (200) in a secondinstance. The cells on both substrates are allowed to incubate for asufficient period of time (according to the cell type and cultureconditions) in the presence of the test compound. The number of cells onthe first region (300) is then determined. The present inventioncontemplates that by comparing results obtained from substrates havingtest compound added to the media and substrates having test compoundadded only to the reservoir (300), a determination can be made as towhether the test compound is chemotactic or chemokinetic. Chemokineticactivity will result in a net increase in cell number in both regions300 and 400 region while chemotaxis will cause a marked increase(greater than in the case of chemokinesis) in cell number in the regionclosest to the reservoir release point of the test compound (300) and anegative chemotactic effect will cause an increase in cell number in thetest region further away from the reservoir source (400) with aconcomitant decrease in the patterned region that aligns liquidcrystals(300). In preferred embodiments, appropriate controls of mediaonly and media plus fetal bovine serum (FBS) are also run.

In still further embodiments, the substrate described in FIG. 16 ismodified so that the entire substrate (100) is configured to orientliquid crystal (See FIGS. 17, 18, and 19). In these embodiments, cellsare seeded on the cell seeding region (500) and a test compound added tothe reservoir (400). A test compound is chemotactic if the cells (600)are stimulated to move out of the cell seeding region and show apreferred orientation in migration towards the gradient of the stimulus.(See, FIG. 17). This effect can be determined by determining the cellnumber present in the first test region (200) and/or the second testregion (300). The test regions do not have any differing physicalcharacteristics. They are simply the zones used for data acquisition andmay be configured to correspond to regions read out by commerciallyavailable plate readers.

A test compound is chemokinetic if the ratio of cells in the firstregion (100) to those in the second region (200) is close to 1, thusindicating the cells moved out of the cell seeding area but showed nopreferred orientation in migration despite the gradient of the stimulus.(See, FIG. 18). A test compound that fails to induce any, or only a veryfew, cells to enter either Field A (200) or Field B (300) has little orno affect on cellular migration. (See, FIG. 19).

If a cell seeding region (500) is not incorporated into the design thencells uniformly distributed across the first (200) and second (300) testregions will not show a significant difference in cell number in thecase of a compound that is purely chemokinetic (that is, does notpromote proliferation). The use of such a design (i.e., cells evenlydistributed rather than limited to a specific cell seeding region) wouldallow the determination of positive and negative chemotaxis but wouldnot distinguish between no effect and positive chemokinesis.

In still other embodiments, the present invention provides assaymaterials and methods for determining the interactions of multiple testcompounds on cell motility and migration. FIGS. 20A-C shows onecontemplated assay configuration for determining the effects andpotential interactions of three test compounds on cell migration.Briefly, FIGS. 20A-C provide a suitable substrate (100) having aplurality of assay regions (200) each providing a localized region ofordered substrate. In the embodiment depicted in FIGS. 20A-C, aplurality of assay regions (200) are arranged in a concentric circlearound the perimeter of substrate (100), the present invention is notintended to be limited however to this configuration. A number ofalternative arrangements are contemplated where the effects of themultiple test compounds can be determined (e.g., rows and columns ofassay regions arrayed on substrate (100), or bands of assay regionsradiating from a center point on substrate (100), etc.). In otherembodiments, two or more types (e.g., local regions of orderedsubstrate, and/or local regions of disordered substrate) of assayregions are provided on a substrate. In a preferred embodiment, each ofthe assay regions (200) represent a zone of structured substrate surfacethat allows for the alignment of liquid crystals except where cells arelocated. In preferred embodiments, the various test compounds areprovided on substrate (100) in test compound positions (301-304) asindicted in FIGS. 20A-C. In certain of these embodiments, each ofpositions (301-304) is a separate reservoir. The present invention isnot intended to be limited however by the number or arrangement ofpositions (301-304) as currently shown in FIGS. 20A-C. Indeed, in someembodiments, greater or fewer than four positions are provided, and/orthe arrangement of the positions on substrate (100) is altered (e.g.,rows and, or positions radiating from a center point on substrate (100),etc.). In the example provide in FIGS. 20A-C, position 301 is contactedwith test compound A, position (302) is contacted with test compound B,position (303) is again contacted with test compound A, and position(304) is contacted with test compound C. In the embodiment depicted inFIGS. 20A-C, substrate (100) also provides a cell seeding region (e.g.,a depression) (400) for seeding cells. Additional embodiments providefor alternative arrangements of one or more positions for seeding cells(400). Additionally, it is possible to uniformly seed cells across thetest substrate (100) encompassing all test regions (200). Afterincubation, (time dependent on cell type, cell line and cultureconditions) positive chemotactic effects will be manifested byaccumulation of cells in proximity to factors (positions 301-304) havinga positive effect.

One contemplated cell migration assay protocol using the substrate shownin FIG. 20A is described below. Briefly, the cells are seeded atposition (400) and allowed to attach for a suitable period of time(depending on cell type and culture conditions) usually from 1-24 hrs.After this attachment period, cell media containing test compounds areplaced at positions (301-304). An additional incubation period(determined by cell type and culture conditions) is provided to allowfor the seeded cells to be contacted by the test compounds. The numberof cells present in each of the assay regions (200) is determined byusing the methods and compositions described herein. For example, if atest compound acted alone and failed to display synergism with othercompounds, then the greatest number of cells would be present in theassay region (200) directly aligned with the position (301-304) wherethat compound was respectively contacted to the substrate (100). If apositive interaction occurs between test compound A (positions 301 and303) and either of test compounds B (position 302) or C (position 304)then the greatest number of cells would be located at positionsintermediate between the respective positions (301-304) where these testcompounds were contacted to substrate (100). FIG. 20B demonstrates apositive chemotactic effect for the test compound located at position302 (Compound B) that is manifested by an increased number of cellsbeing located in the test region (200) in closest proximity to testcompound reservoir position 302. FIG. 20C demonstrates the anticipatedresults from a synergistic interaction on cell migration being observedbetween two different test compound (Factor A positions 301 and 303 andFactor B position 302).

In other embodiments, substrates are engineered to provide from one ormore assay zones. In some of these embodiments, a single substrate isfabricated to provide multiple distinct assay zones to allow for runningreplicate determinations with multiple controls. In preferredembodiments, each of these assay zones is configured to correspond tothe substrates depicted in FIGS. 11-20 (e.g., to comprise a plurality ofthe substrate formats described above). In particularly preferredembodiments, these substrates are configured for use with commerciallyavailable plate readers (e.g., assay substrates are engineered toprovide distinct zones that spatially correspond to test positionsrecognized by commercial plate readers [e.g., 24, 96, 384, 1536 wellplate formats]). In further preferred embodiments, assay substrates areprovided that are configured to be inserted into wells of commercialplates and plate readers.

FIG. 21 shows one embodiment where the disclosed assay substrates areengineered for compatibility with commercially available 24 well platereaders. FIG. 21 represents a substrate comprising a standard 6×4arrangement of 24 individual assay surfaces.

Briefly, in each of the 24 assay surfaces represented, an assay region(100) and a reservoir (200) for contacting a various test compounds ofinterest. In preferred particularly embodiments, the plate configurationdepicted in FIG. 21 utilizes a reservoir system, described in greaterdetail herein, that allows for differentiating chemotactic andchemokinetic cell migration. In yet another embodiment, FIG. 22 shows anadaptation of the assay plate configuration shown in FIG. 21, whereineach of the individual assay zones further provides a cell seedingregion (400) (e.g., depression) for seeding cells.

Additional embodiments provide assay substrates designed to be read bycommercially available plate readers (e.g., 24, 96, 384, 1536 wellformats) that employ a single substrate surface and single reservoir.For example, FIG. 23 shows one contemplated embodiment where a singlesubstrate surface (100) is engineered to provide an array of frombetween 24 to 1536 assay regions (200). In some of these embodiments,the substrate (100) is further configured to provide one or more testcompound reservoirs (300).

In some embodiments, the substrates shown in FIG. 23 are utilized byseeding cells evenly across the across a first and a second substrate.The user then allows the seeded cells a sufficient amount of time(depending on cell type and culture media), usually from 1-24 hours, toattach to the respective substrates. The first substrate is contactedwith a test compound added to the cell media. The second substrate iscontacted with a test compound added to the reservoir (300). Therespective substrates are then incubated for an appropriate period oftime (depending on the cell line and culture conditions) to allow thetest compounds to act on the seeded cells. At the end of the incubationperiod, the media covering the cells is removed and a thin film ofliquid crystal is placed on the surface of the substrate with attachedcells. The number of cells in each of the assay regions of therespective substrates is then determined by visual inspection or byusing an automated plate reader. If a compound is chemotactic, then theassay regions (200) closer to the reservoir (300) would have increasednumber of cells and the assay regions (200) further from the reservoir(300) would have decreased number of cells when compared to controlsubstrates (100) and compared to assays in which the test factor(s) havebeen added to the media covering the cells.

In still another embodiment, the present invention provides assaymaterials for determining the effect on cell migration of multiple testcompounds on a single substrate. In preferred embodiments, thesesubstrates are engineered to have size and shape (e.g., 24, 96, 384,1536, well format) compatible with commercially available plate readers.FIG. 24 shows one contemplated multiple test compound assay substrate.The assay substrate (100) shown in FIG. 21, provides 24 distinct assayregions (200). In the format depicted in FIG. 24, the assay systemallows for the testing of up to 12 different compounds of interest at atime. The present invention is not intended to be limited however to a24 well format. Indeed, other embodiments of the present inventionprovide from 96, 384, and 1536 well formats, these formats are easilyadaptable to commercial plate readers and would provide, respectively,for the simultaneous testing of from 48, 192, to 768 compounds ofinterest. Briefly, FIG. 24 represents a multiple assay region formattedassay plate, wherein each of the 12 test zones (100) comprises twosuitable assay regions (200 and 210), an optional cell seeding region(300) (e.g., a depression) for seeding cells, and a reservoir (400) forcontacting a test compound of interest to the substrate (100). Incertain embodiments, the assay regions (200) provide a localized area ofordered substrate that can anchor liquid crystals. In other embodiments,the entire plate is engineered to provide a suitable assay substrate(e.g., the entire surface can anchor liquid crystals. In someembodiments, the assay regions (200 and 210) are discreet zones, whilein other embodiments, the assay regions (200 and 210) are simply thearea that will be read by the plate reader. A test zone can compriseone, two, or more areas that are read by a plate reader. In FIG. 24, thetest zone (100) comprises two regions (200 and 210) that are read by aplate reader. The present invention contemplates that the configurationshown in FIG. 24 is particularly well suited for differentiating theeffects on cellular migration of multiple test compounds (e.g.,chemotaxis versus chemokinesis).

FIG. 24 depicts use of a 24 well format that allows the running of 12distinct assays on a single plate. This design allows for separation ofchemotaxis from chemokinesis. In FIG. 24 it can be seen that cells canbe seeded in region 300 and allowed to attach. Test compound orappropriate control media is placed into the designated reservoirregions (400). If a compound is chemokinetic, there will be astimulation of cell migration but no directional preference will beevidenced by the cells and both regions 200 and 210 will evidence highercell numbers compared to appropriate controls. If a test compound ischemotactic the increase in cell numbers will be greatest in the readoutregion closest to the reservoir (200) surpassing that found in theregion further away from the reservoir (210).

In some embodiments, the substrate shown in FIG. 24 is utilized byevenly seeding cells in each region (that in FIG. 24 are illustrated asbeing restricted to the cell seeding positions) (300) across the assayplate (100) The uniformly distributed cells are allowed a sufficientamount of time (depending on cell type and culture conditions) to attachto the substrate (100). Test compound or appropriate control media isthen added to reservoirs (400). After an appropriate incubation time(e.g., determined by cell type and culture conditions) the plate isread. In operation, a test compound is added to the reservoirs in thewells. If zone A (position 200) consistently indicates a greater numberof cells than zone B (position 210), then a chemotactic effect of thetest compound is indicated. It will be recognized that these plateformats find use with other biophotonic detection techniques, includingfluorimetric, calorimetric, and densitometric techniques.

In still other embodiments of the present invention, the substrates areengineered to include two or more microfluidic channels for producinguniform gradients of one or more test compounds over a plurality ofassay regions. For example, FIG. 25, shows one contemplated embodimentof present invention that employs three microfluidic channels that allowfor the simultaneous testing of three different compounds of interest ona single assay plate with a plurality of assay regions for eachcompound. Briefly, FIG. 25 shows a 48 well assay plate which has beendivided into top, middle, and bottom panels, A, B, and C respectively.Each of panels A, B, and C presents 8 individual cell motility andmigration test regions on a single assay plate. Thus, in preferredembodiments, the effects of each of three test compounds on cellularmotility and migration can be probed in octuplicate. The presentinvention is not intended to be limited however to the assayconfiguration presented in FIG. 25. Indeed, the present inventioncontemplates embodiments of multiples of other commercially recognizedwell formats. For example, the present invention specificallycontemplates embodiments using a 96 well assay plate format. Such anassay format would allow for the simultaneous testing of 8 testcompounds in dodecatuplicate, or 12 test compounds in octotuplicate,etc. In regard to panel A of FIG. 25, each test region (100) comprises,two suitably ordered assay regions (200 and 210) and a cell seedingregion (e.g., a depression) for seeding cells (300), and a network ofreservoirs and microfluidic channels (400) configured to deliver a testcompound to the test regions. The network (400) comprises a mainreservoir (410) in fluidic contact with a plurality of test zonereservoirs (e.g. 420 and 440) via communicating microfluidic channels(e.g. 430). In some embodiments, the assay regions (200 and 210) arediscreet zones, while in other embodiments, the assay regions (200 and210) are simply the areas that will be read by the plate reader. A testregion can comprise one, two, or more areas that are read by a platereader. Referring to FIG. 25, the assay regions (200) are aligned invertical columns A and B for each replicate (100) shown. In preferredembodiments, a reservoir and microfluidic channel (400) horizontallybisect each panel and form the left edge of each replicate. It can alsobe appreciated that the flexibility of this design allows for thetesting of a single compound or of multiple compounds simultaneously ifadmixed in the initial reservoir (410) or supplied in differentreservoirs in separate plate designs (e.g., see FIG. 26).

One contemplated cell migration and motility assay using the assay plateand shown in FIG. 25 is described below. Briefly, cells are seeded intoeach cell seeding region (300) for panels A, B, and C. The cells areallowed a sufficient period of time (depending upon the cell type andthe culture conditions) to attach to respective positions (300). Adifferent (or identical) test compound is added to each of the mainreservoirs (410) in panels A, B, and C, respectively, so that the testcompound is delivered via the network to each test region reservoir(e.g., 420 and 440). The respective test compounds are allowed asufficient period of time (depending upon cell type and cultureconditions) to act on the cells in each test region. Each test region isanalyzed (e.g., using a suitably modified commercial plate reader andappropriate software or visually with a microscope) to determine(“read”) the number of cells in each replicate that migrated into assayzones aligned in columns A and B, respectively. In preferredembodiments, the “reading” step is performed by overlaying the substratewith liquid crystals as described in detail above. In some embodimentsthis plate design and principles are used in conjunction withcalorimetric and fluorimetric plate readers using standard stainingprotocols for imaging of cells or of indirect indicators of cellviability such as intrinsic enzyme activity.

In still other embodiments of the present invention, assays are providedfor determining inhibitory, additive, or synergistic effects of testcompounds on cellular motility and migration. For example, in regard tothe preferred embodiment shown in FIG. 26, a suitably ordered assayplate (100) is configured with an array of assay regions (400 and 500)arranged to be compatible with standard commercial plate readers, andtwo or more separate reservoirs (200 and 300). Referring to FIG. 26, itcan be seen that this embodiment is designed to provide 3 distinct testzones labeled zone A, zone B and zone C. In use, a first compound knownto be chemotactic for a particular cell type is placed in one of thereservoirs (200) and a second test compound is added to the other of thereservoirs (300). This plate design allows the evaluation of two testcompounds and comparison to control media. Cells are seeded on thesubstrate in optional cell seeding areas (600) and allowed to attach asdescribed in the embodiments above. Test compound A is placed in testreservoir (300) in zone A, test compound B is placed in test reservoir(300) in zone B and control media is placed in test reservoir (300) inzone C. Communicating microfluidic channels provide the knownchemotactic agent (210) and test compound (310) to local test regionreservoirs that deliver these agents to the cells on the test substratein a gradient. Presence of cells in the assay regions (400 and 500) ispreferably assayed by overlaying the substrate with liquid crystals asdescribed above. A simple determination of the effects of the testcompound can be made by comparing the results with control assays. Theknown chemotactic agent will stimulate cells to move into the assayregion closest to the local reservoirs containing this compound (210).If a test compound (in zones A and B, deposited into reservoir 300 anddelivered to local reservoirs 310) is able to inhibit chemotaxis incitedby a known chemotactic agent then fewer cells will be localized to thetest region closest to the test reservoirs (500) compared to controls(in zone C). Additionally, if a test agent is synergistic or additive instimulating chemotaxis then more cells will be located in zone 500compared to controls. If a compound completely inhibits migration ofcells in the presence of a known chemotactic agent then fewer cells willbe located in both assay regions 500 and 600 compared to controls. Incertain embodiments it is anticipated that this design will be used inconjunction with colorimetric and fluorimetric assays. In preferredembodiments the detection of cell number will employ placing a thin filmof liquid crystal overt the test substrate (with assay zones that alignliquid crystal mesogens) and any attached cells. As previouslydescribed, the presence of cells will mask the nanostructured substratelocally thus prevent the LC from gaining access to the orderinginfluence of the substrate. In preferred embodiments, this is detectedand quantified using polarized light, specific wavelengths orcombinations of wavelengths of light.

Additional embodiments use this design of multiple reservoirs andmicrofluidic channels to create separate test zones for use in cellinvasion assays described previously (See, e.g., FIGS. 5-9). This allowsfor the determination of the ability of a test compound to prevent cellinvasion, which is often used as an indication of probable therapeuticefficacy in the development of anti-neoplastic compounds.

Additional embodiments and variations of the disclosed assayconfigurations are within the scope of the present invention. In someembodiments, the cells seeded onto the assay substrates described hereinare labeled on their surfaces with one or more fluorescent molecules,radioisotopes, and the like. It is contemplated that in someembodiments, labeling will increase the detectability and sensitivity ofthe signals generated by the disclosed assay systems. For example, incertain embodiments, a standard commercial plate reader (adapted tohandle the LC assay formats described herein) is used to analyze areasof disorder and order in a liquid crystal and then determine cell numberby analyzing fluorescence. The art is well acquainted with cell labelingtechnologies and with detecting labeled cells.

In other preferred embodiments of the present invention, asymmetrically(e.g., partially transparent and partially opaque) patterned substratesare provided that are useful for conducting cell migration assays thatemploy liquid crystal, fluorometric, calorimetric or densitometricreadout methods. In the liquid crystal assay embodiments, theasymmetrically patterned substrates are configured to provide regions oforder that orientate mesogens or to provide regions of disorder (e.g.,random patterning) that do not orientate mesogens. The present inventioncontemplates that certain embodiments employing asymmetric substratesare useful for determining the effects of a test compound on cellmigration by providing a tool for distinguishing chemokinetic fromchemotactic cell migration. The underlying principle behind theembodiments employing asymmetric substrates and the various liquidcrystal based assay configurations disclosed herein is basicallyidentical. However, fluorometric, calorimetric, or densitometric basedmethods find use in these embodiments when the cells of interest arevitally, calorimetrically (e.g., for assays of dead cells) orfluorescently stained or otherwise labeled when the substrate providedis asymmetrically patterned with both optically transparent andoptically opaque regions.

In preferred embodiments, cells (e.g., at least one cell) are seeded onthe optically opaque portion of the assay substrate. In some of theseembodiments, the cells are seeded in a cell seeding region (e.g., adepression in the assay substrate). In other embodiments, the cells aresimply evenly distributed across the substrate and allowed sufficienttime under appropriate conditions for attachment. The test region,encompassing both optically transparent and optically opaque (orpatterned and non-patterned regions; the patterned regions of whichorient LC mesogens) can be configured to correspond to the assay regionsread out by commercial plate readers.

In certain embodiments disclosed herein, test compounds are placed inone or more reservoirs and the assays are conducted using protocolssimilar to those used in the liquid crystal based assays describedabove. In other embodiments, the asymmetrically patterned assaysubstrate further provides at least one microfluidic channel fluidicallyconnecting at least one reservoir and the optically opaque and theoptically transparent regions of the substrate.

The present invention contemplates detecting cells that migrate onto thetransparent regions, or regions that are patterned to align liquidcrystals of the asymmetric substrate. Cells that migrate onto the opaqueregions or regions that don't align liquid crystals of the asymmetricsubstrate are hidden from detection.

FIG. 27 shows one embodiment of a contemplated asymmetric assayconfiguration. Referring to FIG. 27, Panel A depicts the use of anasymmetrically patterned nanostructured substrate for use with liquidcrystal assays and Panel B depicts the design of an asymmetricallyopaque substrate for use with calorimetric, fluorimetric, radiometricand other assays that detect the presence of cells. The assay substratecomprises a first region (100) which is patterned to align liquidcrystal mesogens (panel A) or is substantially transparent for use withother assays of cell detection (panel B) and a second region (200) thatdoes not orient liquid crystals and thus will not report the presence ofcells with liquid crystal assays (Panel A) or is substantially opaqueand will not report the presence of cells using other assays(exemplified by but not limited to calorimetric and fluorimetric assays)of cell detection (panel B). In certain embodiments, at the center ofregions (100) and (200) is a position (e.g., depression) provided forseeding cells (300). The regions in FIG. 27 are depicted as semicircles.However, it will be understood that the substantially transparent andsubstantially opaque regions may be different in size and shapedepending on the nature of the assay and detection equipment used. Insome embodiments, protocols for using asymmetric embodiments of thepresent invention are substantially the same as those for using theconventional LC assay configurations described above.

FIG. 28 shows the expected effects of a test compound that exerts achemotactic effect on cell migration. The arrows (100) indicate that achemotactic gradient is originating from the left hand side. Panel Adepicts the use of asymmetric substrates for use with liquid crystalassays. The presence of cells is only reported when they are located onregions that are nanotextured as to align liquid crystal mesogens (lefthand side of substrate A in panel A and right hand side of substrate Bin panel A). It is contemplated that by using paired asymmetricallypatterned substrates that are oriented differently (see substrates A vs.substrates B in Panels A and B) in regards to the direction of achemotactic signal and comparing the results, the user of the device isable to sort out whether a given test compound has no effect, or whetherit induces chemotaxis or chemokinesis. With chemotactic stimuli, thecells move towards the origin of the chemotactic signal (100). A markedincrease in the number of cells is detected when the patterned substrateis oriented on the left (substrate A of panel A).

Panel B of FIG. 28 demonstrates the use of asymmetrically patternedsubstrates for use with calorimetric or fluorimetric plate readers ofother imaging devices that assay for cell number. Similar to the liquidcrystal assay substrates, only one-half of the substrate allows for thedetermination of cell number (the left hand portion of substrate A andthe right hand portion of substrate B in panel B).

FIG. 29 demonstrates the anticipated results for a test compound thatstimulates chemokinesis (e.g., by demonstrating an increase in cellmigration without a directional vector as regards the stimulus). Thetest compound gradient is originating from the left hand side (100).Panel A (substrates A and B) illustrates the results expected withliquid crystal assays and Panel B illustrates the results expected usingcolorimetric or fluorimetric assays. A greater number of cells aredetected compared to controls (e.g., no chemotactic stimulus provided)and an equivalent number of cells are detected regardless of whether thesubstrate portion that can detect the presence of cells is oriented onthe left (substrates A) or on the right (substrates B) relative to thedirection of test compound gradient (100—originating from the left).

It is important to note that the asymmetric substrate embodimentspresent one adaptation of the general schemes and embodiments disclosedherein for conducting cell migration and motility assays. Thus, thepresent invention specifically contemplates that the substrates maycomprise one or more microchannels and one or more reservoirs asdescribed in detail above. Similarly, asymmetrically patternedembodiments are equally adaptable to multiple assay region microarrayand plate reader sizes and formats (e.g., conforming to standard 24, 96,384, 1536 well plate reader formats).

In some preferred embodiments, the assays comprise a plurality (e.g., 2,4, 12, 24, 48, 96 or more) of asymmetrically patterned regionsorientated in an array. In particularly preferred embodiments, adjacent(e.g., neighboring) asymmetric substrate regions are arranged to providealternating (e.g., opposing) orientations for neighboring opticallyopaque and the optically transparent regions. FIGS. 30 through 32illustrate several contemplated asymmetrically patterned assayconfigurations designed to determine the affects of a test compound (orseveral test compounds and known compounds) on cell migration.

Referring to FIG. 30, a plurality of asymmetrically patterned assayregions (200) and (300) are configured in an array format on substrate(100). While illustrations CC through EE depict the use ofasymmetrically patterned substrates for use with liquid crystal assays,the principle of asymmetric sampling of a substrate surface in amultiarray format is applicable also for use with calorimetric orfluorimetric assays as detailed in FIGS. 27 through 29. Eachasymmetrically patterned assay region (200) and (300) further provides aregion that is patterned to align liquid crystal mesogens (400) and aplanar region that does not align liquid crystal mesogens (500). Thepresence of cells would only be reported, after the addition of liquidcrystal mesogens, on assay regions that are capable of aligning themesogens. The neighboring asymmetrically patterned assay regions ((200)and (300)) are orientated in a opposing arrangement in regard to therespective planar regions (400) and patterned regions (500) and locatedorthogonally relative to the site of origin of a test compound gradient(700). In preferred embodiments, the patterned region (500) of eachasymmetrically patterned assay region ((200) and (300)) provides a cellseeding position (e.g., a depression) (600). The embodiment depicted inFIG. 30 further provides a reservoir (700) at one edge of substrate(100) for holding and dispersing at least one test compound.

It is contemplated that the assay configuration illustrated in FIG. 30provides an increasingly attenuated concentration (dashed lines) of testcompound from reservoir (700) to the asymmetrically patterned assayregions ((200) and (300)) as the distance of the asymmetricallypatterned assay regions ((200) and (300)) from reservoir (700)increases. Analysis of cell migration data obtained from asymmetricallypatterned assay regions ((200) and (300)) provides information as to therelative potency of the test compound. For instance, a stronglychemotactic test compound more strongly stimulates the migration ofcells towards the origin of the compound (700) in the asymmetricallypatterned assay regions ((200) and (300)) distant to reservoir (700) ata given time point than would a weakly chemotactic test compound. Inpreferred embodiments, the asymmetrically patterned assay regions ((200)and (300)) are orientated on substrate (100) to provide for automateddata acquisition and analysis (e.g., using a plate reader).

FIG. 31 represents another embodiment where substrate (100) provides oneor more rows that serve as controls of asymmetrically patterned assayregions (800) that are prohibited from contacting a test compound. FIGS.31 and 32 also illustrate assay configurations where one or moremicrofluidic channels. (e.g., 900 of FIG. 31 and 750 & 850 of FIG. 32)are provided to uniform delivery of test compounds across the substrate(100).

The present invention is not intended to be limited to theasymmetrically patterned assay configurations described above and inFIGS. 30 and 31. Indeed, a number of assay region configurations arepossible when employing larger numbers (e.g., 48, 64, 96, 384, or 1536,or more) of asymmetrically patterned assay regions on a singlesubstrate. FIG. 32 briefly illustrates an embodiment where a firstreservoir (700) and accompanying microfluidic channels (750) containinga known chemotactic agent are positioned in proximity to a secondreservoir (800), and accompanying microfluidic channels (850) containinga test compound. The other elements illustrated in the asymmetric assayconfiguration depicted in FIG. 32 are common to those described in FIGS.30 and 31, respectively. It is contemplated that the model assayconfiguration illustrated in FIG. 32 provides for evaluating the abilityof a test compound to affect (e.g., augment or inhibit) a knownchemotactic agent.

In some embodiments of the use of asymmetric substrates with patternedsurfaces, the assay region will be patterned over its entire surface,but asymmetric sampling will occur due to the asymmetric presence of anunderlying opaque component that prevents the transmission of light.FIG. 33 demonstrates one such embodiment. In FIG. 33, panel A is atop-view of an assay region (100) and panel B is a cross sectional viewof the same assay region. It can be seen that the assay region isasymmetric in its sampling capabilities, with light being transmittedthrough region 200 but not through region 300. The entire surface of theassay region is nanopatterned (600) to align liquid crystal mesogens.Underlying the nanopatterned surface however is an asymmetrically opaquesubstrate that is optically clear (400) underlying region 200 andoptically opaque (500) underlying region 300. Thus, data are collected,as to the presence of cells on the surface (which disturb theorientation of liquid crystal mesogens placed on their surface) only inthe portion of the substrate that is optically clear (200). This designhas application to all of the embodiments described above that utilizeasymmetrically patterned or asymmetrically opaque substrates (e.g. seeFIGS. 27 through 32)

It is possible to use this strategy by programming a plate reader totake asymmetric readings within a single test area. Currently, manycommercial plate readers take multiple readings within a single well andaverage the readings. A reader could be programmed to take readingsasymmetrically within a test area and could therefore be used todetermine chemotaxis and chemokinesis as outlined in previousdescriptions. These embodiments are illustrated in FIG. 34.

In some preferred embodiments, a plate reader is programmed so that thesensing element(s) obtain multiple discreet readings within a singleanalytic zone or to scan an analytic zone to locate the perimeter andcalculate the diameter of an outwardly spreading population of cells. Inthis embodiment, cells are seeded centrally onto a distinct cell seedingarea. In some embodiments, the cells are seeded using a specificallydesigned cell seeding device described below that constrains thedistribution of cells to a discrete location within the analytic zone(such as the center of a single well of a 24,96,384 or 1536 multiwellplate). After initial incubation in the cell seeding device to allow forcell attachment (time dependent on culture conditions and cell type),the cell seeding device is removed and nonattached cells gently removedby irrigation. The substrate with cells is then incubated again (timedependent on culture conditions and cell type) and the migration ofcells outward from the initial cell seeding area determined using theprogrammed sensing elements and analytic software. If a factor is addedto the media (such that no gradient of the factor is present in themedia) that promotes cell migration then the outline of the outwardlymigrating cells will roughly define a circle with the diameter of thecircle being proportional to the potency of the factor to stimulate cellmigration.

Similarly, this procedure could be used to evaluate compounds thatinhibit cell migration. Such assays are important for screening ofpossible therapeutic compounds for the treatment of cancer. In somepreferred embodiments, this procedure is used to assess the ability oftest compounds to inhibit migration of vascular endothelial cells whichis often used as a positive indicator for compounds used in cancertreatment. The programming of the mechanical drivers of the sensingelement(s) and analytic software is easily accomplished by those ofordinary skill in the art. In some embodiments, the cell seeding area isoptically opaque to avoid being read out as containing cells or it couldbe transparent and the perimeter of the cells seeded into a defined arearecorded and their location determined. In other embodiments, thesensing element(s) are programmed to avoid obtaining readings from theinitial cell seeding area.

It must be noted that this approach is not limited to use with liquidcrystal based assays but is broadly applicable to biophotonic techniquessuch as colorimetric, optical densitometric, fluorometric andradiometric methodologies. Using this strategy, assays that separate outchemokinesis from chemotaxis can be performed by provision of achemotactic gradient to the initially seeded cells (using a fluidicdelivery system or a degradable pellet to create a gradient ofchemotactic factor(s)) and then obtaining multiple readings of celllocation within an analytic zone. The software provides either analysisof discrete regions within an analytic zone or is used to determine theresultant size and major axis of the dispersed colony of cells. If acompound is chemokinetic the dispersed cells will be evenly distributedaround the initial cell seeding area creating a roughly circular outlinewith the diameter of the circle being proportional to the potency of thechemokinetic compound while a chemotactic compound will induce the cellsto preferentially migrate towards the source of the factor creating anellipse with the long axis diameter being proportional to the relativechemotactic potency of the compound. This procedure could be done bymodifying the software that determines the spatial location of thesensing element(s) and the analytic software. In another embodiment ofthe invention, a motive force (including but not limited to electricalfields, magnetic fields, and thermal fields) is used to orient thinfilms of liquid crystal mesogens such that they report the presence andspatial location of cells attached to a substrate. In this embodiment,the mesogens are not in direct contact with the cells. This embodimentmakes use of the fact that attached cells create defined zones ofimpedence (e.g., electrical and/or magnetic and/or thermal) thatattenuates the motive force(s) transmitted into the liquid crystal film.An example of this embodiment is depicted in FIG. 35. Cells (100) areseeded onto a non-conducting substrate (200) and allowed to attach (theattachment time is dependent on cell type and culture conditions). Thesubstrate (200) may be functionalized by adsorption or covalent bondingof constitutents that support cell attachment and function (including,but not limited to, extracellular matrix proteins such as collagens,laminins, fibronectin, vitronectin, osteopontin, thromospondin,Intercellular adhesion molecule-1 (ICAM-1), ICAM-2, proteoglycans suchas chondroitin sulfate, von Willebrand factor, entactin, fibrinogen,tenascin, Mucosal adressin cell adhesion molecule (MAdCAM-1), C3b, andMDC (metalloprotease/disintegrin/cysteine-rich) proteins), nucleicacids, specific receptors and cell receptor recognition sequences (e.g.,cadherein, immunoglobulin superfamily, selectin, mucin and integrinbinding sequences such as RGD, EILDV, LDV, LDVP, IDAP, PHSRN, SLDVP,GRGDAC, and IDSP)). Once attached, a thin film of liquid crystalmesogens (300) is placed in contact with the surface of the substrateopposite to the surface to which cells are attached. With attached cellsit makes no difference if the cells are on the upper or the lowersurface. A motive force (400, represented by large arrows on lower partof diagram) that is capable of orienting liquid crystal mesogens once athreshold level is attained, is applied across the substrate (100). Themagnitude of the motive force is set to be just above threshold fororienting the liquid crystal mesogens overlying the substrate (200) inregions where cells are not attached. At near threshold values, theimpedence of attached cells will prevent the orientation of mesogenslocated on the substrate (200) immediately opposite their site ofattachment. The diferential attenuation of the motive force as it passesthrough the cells, substrate and subsequently through the LC film isrepresented by differing size arrows in the upper part of the diagram(500). This results in regions of the liquid crystal film where mesogensare oriented by the motive force and regions (correlating to cellattachment zones) where mesogens are not exposed to a sufficientmagnitude of motive force to cause orientation of mesogens.

In preferred embodiments, the motive force is an electric field appliedby first and second electrodes positioned on either side of thesubstrate. In preferred embodiments, an aqueous electrolyte isincorporated between the cell side of the substrate and the first orsecond electrode. The electrodes are preferably either solid slab-typeelectrodes, or more preferably made of a wire mesh. In operation, apotential difference is applied across the substrate. The presence ofcells perturbs the electric field lines in a manner that perturbsorientation of the liquid crystal, causing the cells to be imaged.

This embodiment has applications for the detection of cell number usefulin assays of cell attachment and cell proliferation. Additionally, sincethe magnitude of motive force impedence directly correlates with firmcell attachment to the substrate, this embodiment is useful forevaluating the toxic effects of test compounds. A compound toxic tocells will cause weakening or dissolution of attachment to theunderlying substrate that in turn results in a quantifiable loss ofimpedence thus making the mesogens opposite the region occupied by thecell more succeptible to the orienting influence of the motive force(s).

FIG. 36 demonstrates another embodiment of the invention. In thisembodiment, cells (100) are seeded on a substrate (200) that has 10 nmto 1 μm diameter perforations in it (300). Such a substrate can befabricated using a variety of techniques including but not limited tophotolithography, x-ray lithography and e-beam lithography. Theregistered array of perforations would be introduced into a substratethat is insulating in nature, preventing transmission of motive forcesinto the liquid crystal mesogens located on the surface opposite thesurface cells are attached to. A thin conducting fluid layer(biologically inert to cells) is placed on the surface opposite thecells prior to placement of a thin film of LC mesogens. This createsdistinct zones of readout of known spatial distribution that willfacilitate development of automated process for information processing.

In other embodiments, cells are grown to confluence on test substrateswhich allow assessment of the integrity of intercellular adhesions.Similar to assays that utilize transepithelia membrane resistance to theconduction of electric fields, this assay has applications to studies offunctional morphology, for studies of pathobiology of infectiousorganisms, to the evaluation of toxic effects of test compounds, and tothe in vitro evaluation of neoplastic invasiveness (Zak et al). Anexample of this embodiment is given in FIGS. 37A and 37B. Cells (100)are seeded on a substrate (200) that conducts a motive force (eg,electrical and/or magnetic and/or thermal fields). Epithelial cells areincubated and allowed to grow to confluence at which point they developintercellular junctions (300) which makes them more resistant totransmitting motive forces through the substrate and subsequently into athin film of liquid crystal mesogens (400). At near threshold values ofmotive force (large arrows in lower part of illustration (500) theintact cellular monolayer prevents transmission of sufficient motiveforce to align LC mesogens. The attenuation of motive force by theintact epithelial monolayer is depicted by small arrows (600) in theupper part of FIG. 37A.

FIG. 37B depicts the consequences of exposure to a test compound that istoxic for the epithelial cells. In this case cellular toxicity (100) ismanifested by a breakdown of the intercellular junctions (300) and aloss of impedance (resistance) of the epithelial layer to transmissionof motive force(s) into the mesogenic layer located on the surfaceopposite the cells. In this case, the motive force is sufficient(depicted by large arrows—600) to align the mesogenic layer. This changein the orientation of the liquid crystal mesogens can be quantitated byall of the means previously described in this application. Orientationshifts in the liquid crystal mesogen orientation can be continuouslymonitored prior to and after exposure to test compound or can bequantitated at pre-determined time points.

In some embodiments, the substrate (100) consists of an insulatingmaterial (200) (e.g., glass in the case of using electric fields as themotive force) with a central window (300) comprised of a conductingmaterial, capable of supporting cell attachment and function, thatallows the transmission of motive force(s). These relationships aredepicted in FIG. 38A.

FIG. 38B illustrates the use of this substrate with cells. Cells (400)are seeded on the substrate (100) that is comprised of an insulatingmaterial (200) and contains a central window (300) that is conductivefor the motive force(s) being used to orient LC mesogens. Onceconfluence is reached mature intercellular adhesions form (500) thatcontributes to the resistance to passage of the motive force(s).

In another embodiment, LC mesogens are used to report electricalactivity of cells in culture. Types of cells of interest for studyinclude, but are not limited to, neuronal cells, cardiac cells andmuscle cells. In this embodiment, cells are cultured on standardlaboratory plastic or glassware. A thin film of LC mesogens eithernon-toxic to the cell type being studied and/or separated from the cellby addition of a thin film of phospholipid that has been shown tointerface with mesogenic layers. Electrical activity in the cell resultsin alteration of the orientation of LC mesogens immediately adjacent tothe cell. This change in order can be visualized using all of thetechniques previously described.

C. Cytology Assays

In still further embodiments, the present invention providescompositions and methods for reporting cytoskeletal alignment in cellmembranes. For example, in certain embodiments, liquid crystals are usedto report the order of cytoskeletal elements transmitted through thecell membrane. In some embodiments, the liquid crystal layer is placeddirectly onto the cells of interest. In other embodiments, the membranesof the cells of interest are solubilized (e.g., using surfactants) toreveal the cytoskeleton to the liquid crystal layer itself. In someembodiments, the liquid crystal layer can be the surfactant (e.g.,lyotropic liquid crystals).

In other embodiments of the present invention, compositions and methodsare provided for quantitating the production of specific (ornonspecific) cellular secretory products. The present invention furthercontemplates specific embodiments directed to quantitating the secretionof specific secretory products in response to contacting cells ofinterest with one or more compounds that induce secretion ofextracellular products. In a preferred embodiment, a nanostructuredsubstrate is fabricated with specific binding sequences incorporatedinto its surface (e.g., targeted to bind a specific protein product ofthe cell such as a growth factor or cytokine). Cells of interest areseeded onto the substrate and allowed to attach and grow underappropriate conditions. In a preferred embodiment of the invention, thesurface that supports the cells is a second surface, and the firstsurface is that surface that is fabricated with specific bindingsequences incorporated into its surface. This second surface may beplaced above or below the first surface. Production of a gradient ofliquid crystal response is visualized as a halo effect around thecell(s) with the greatest concentration of a specific factor beingclosest to the cell(s). (See, FIG. 39). Briefly, FIG. 39 shows the zoneof liquid crystal response (200) produced around a cell(s) of interest(100) upon secretion of a specific secretory product by the cell(s). Itis contemplated that an estimation of the amount of product secreted bya cell can be correlated to the diameter (ring) of liquid crystalresponse surrounding the cell. For example, in one specific embodiment,the assays and techniques disclosed herein allow for evaluating cellularsecretion of Nerve Growth Factor (NGF) in response to exposure to one ormore trophic factors such as EGF.

From the above examples, it is clear that the present invention allowsfor the investigation of cellular secretory responses of single cells orsmall groups of cells to a variety of environmental stimuli. It is alsoclear that these techniques and assay substrates allow for the detectionand quantitation of cellular factors that are secreted asymmetrically inresponse to environmental stimuli delivered by additional cells. (See,FIG. 40). FIG. 40 shows, the zone of liquid crystal response (200)produced by a cell(s) of interest (100) upon secretion of a specificsecretory product influenced by the presence of another cell (300).Examples would be the stimulation of secretion of factors by epithelialcells (100) due to factors released by adjacently located neuronal cells(300).

In still other embodiments, the secretory response assays disclosedherein are adapted for use with the substrates, reservoirs, andmicrofluidic systems disclosed above, such that a cell's secretoryprofile can be quantitated in response to one or more compound gradientsestablished across a liquid crystal surface. For example, FIG. 41 showsthe secretory response (300) of two cells (200) reported through asuitable liquid crystal surface (100) in response to a compound gradientestablished by a reservoir (400) on the substrate.

In a preferred embodiment of the invention, micrometer andnanometer-sized channels are patterned into the surfaces that supportthe cells. The flow of liquid along the channels permits the sampling ofsecretory products from the cells that cover the channel. The secretoryproducts collected from the channel can be assayed by using methods thatinvolve the use of liquid crystals, which are well known to thoseskilled in the art, of by conventional methods of analysis such as massspectroscopy, UV-Vis absorption spectroscopy, ELISA, gel electrophoresisand other methods of analysis applicable to secretory products.

It is contemplated that the cell secretory product assays can beengineered (e.g., adapted) for use with any of the cell migration andmotility assay configurations disclosed herein, thus allowing thedetection of asymmetric secretion in association with cell migration. InFIG. 42, the nonspecific interaction of the cell (200) during migrationacross the substrate (100) (having a test compound reservoir (400))creates a change in the order of the surface of the substrate (100) inits wake which is reported by the liquid crystal layer (300), thepresence of the cell (200) is reported by the cell blocking access ofthe ordered substrate to the liquid crystal layer placed on top and thepresence of a specific secretory product (500) of the cell is beingreported by the specific receptors which have been incorporated into thenanostructured surface of the substrate (100). A reservoir (400) hasbeen incorporated into this example to demonstrate an asymmetricsecretory process (500) by the cell (200) in response to a gradient of asoluble factor being delivered across the surface of substrate (100).

Changes in the metabolic states of cells give rise to changes in theheat output. Because the order within a liquid crystalline can be astrong function of temperature, liquid crystals can be used to detectchanges in the heat output of cells. For example, calorimetric changescan be associated with change in metabolic state such as phagocytosisvia stimulation with LPS. This could be by simply placing LC on a singlecell or population of cells on a nanostructured (ordered) substrate orby using the hybrid ECM-LC. A preferred embodiment of the invention,makes use of mixtures of liquid crystals that possess phase transitionsat the temperatures similar to those used for cell cultures. In apreferred embodiment of the invention, the liquid crystal is cooledtowards a phase transition and the appearance of the liquid crystal ismonitored during the ramp in temperature. The liquid crystal can be aliquid crystalline substrate on which cells are grown. A preferredembodiment of the invention is a liquid crystalline substrate that isdecorated with phospholipids or other biological receptors that interactwith cells. The liquid crystals can also be lyotropic liquid crystals,and thus cell culture can occur in the presence of the liquid crystaloverlying the cells during imaging of the cells.

D. Plate Top Devices

The present invention also provides novel plate top devices for use inconjunction with multiwell (i.e., 8, 16, 24, 96, 364 etc.) plates. Insome embodiments, the plate top devices provide anchoring surfaces formesogens utilized in the assay. Referring to FIG. 43, one embodiment ofa plate type device (100) of the present invention comprises a plate topsurface (200). A plurality of elongated members (300) extend outwardlyfrom the plate top surface (200). In preferred embodiments the plate topmembers comprise a distal end (400) having a distal end surface (500).In preferred embodiments, the elongated member (300) is a hollowcylinder. In further preferred embodiments, the distal end (400) iscomprised of an optically clear, non-birefringent material (e.g.,polycarbonate). In further preferred embodiments, the distal end surface(500) is configured to orient mesogens. Any suitable surface preparationmay be used, including, but not limited to, rubbed surfaces, surfacewith obliquely deposited metals, nanoblasted surfaces.

Referring to FIG. 44, the plate top device is placed over a multiwellplate (100, only a portion is shown) having a number of wells (600)equal to the number of elongated members (300) so that each of theelongated members (300) extends into a corresponding well (600).Preferably, the elongated member (300) extends substantially to thebottom of the well (600) so that a thin film of liquid crystal (700) ispresent between the distal end (400) of the elongated member (300). Instill other preferred embodiments, the dimensions of the elongatedmember (300) are only fractionally smaller than the interior dimensionsof the wells (600) so that extra liquid crystal mesogens pass upwardsalong the sides of the elongated members (300) placed in the wells(600). It is contemplated that this arrangement creates uniform contactof the distal end (400) of the elongated member (300) with the liquidcrystal film (700).

The present invention also provides plate tops useful for deliveringcells to a predetermined area in a well of a multiwell plate. Referringto FIG. 45, the cell delivery plate top (100) comprises a plurality ofelongated members (200) extending downward from a plate top (not shown).When the plate top is placed onto a multiwell plate (300) the elongatedmembers extend into the wells (400) of the multiwell plate so that thedistal end (500) of the elongated member (200) contacts the surface(600) of the well (400). In some preferred embodiments, the surface(600) is substantially flat. In other preferred embodiments, the surfaceof the well (600) has a depression therein. Preferably the distal end(500) comprises an opening (550). In some preferred embodiment, theopening is from about 1-4 mm. In some further preferred embodiments, theedges of the opening 550 are coated with a gasket material, ensuring atight seal with the surface (600). Non-limiting examples of gasketmaterial include silicone, latex, and petroleum jelly. In otherembodiments, the edges of the opening (550) are coated with ahydrophobic material to discourage cell seepage or migration beyond theperimiter of the cylinder opening. In preferred embodiments, theelongated member (200) is hollow so that a solution of cells may bepassed through the elongated member (200) to the surface (600). Inparticularly preferred embodiments, the elongated member (200) isconical in shape. However, the invention is not limited to anyparticular shape of elongated member (200). For example, the elongatedmember (200) may be cylindrical and triangular in shape. In furtherpreferred embodiments, the elongated members (200) are oriented withrespect to the wells (400) of the multiwell plate (300) so that cellsare delivered to the center of the wells (400). The plate tops of thepresent invention are not limited to any particular material. Indeed,the plate tops may be formed from stainless steel or tissue culturepolystyrene.

In use, cells in solution are pipetted into the elongated members (200).The cells are allowed to settle and attach to the surface (600) of thewells (200). After being allowed to attach for a specified period oftime (incubation time being determined by cell type and cultureconditions), the plate top is removed and non-adherent cells are removedby gentle washing. Culture media is then added to the wells and astandard plate top is placed onto the multiwell plate. In still furtherembodiments, the plate top devices are used to perform cell invasionassays. In these embodiments, the cells are seeded onto a substrateusing the plate top device (or applied by some other method) and allowedto attach. Non-attached cells are rinsed off and the cells are labeled(e.g., with a fluorochrome or vital dye). The substrate is thenoverlayed with a matrix. The matrix could be an extracellular matrixsuch as a basement membrane like complex collagen 1 or otherextracellular matrices or could be agar. The cells are allowed toincubate. A plate reader is then used as described above in more detailto determine how far the cells have migrated from the original seedingpoint. In other words, the plate reader can be used to identify the areaover which the cells have migrated by detecting the cells labeled cells.This methodology may also be utilized to perform a migration assay,except that no matrix is utilized.

As shown in FIG. 50, in some embodiments the present invention providesa device for facilitating the seeding of cells in a well (100)comprising a plurality of cylinders (105) sized to be inserted into thewells (110) of a multiwell plate (115). In some embodiments, thecylinders (105) are movably connected to at least one horizontal member(120). The device (100) may be positioned over a corresponding multiwellplate (e.g., 115) so that the cylinders (105) extend into the wells(110) and contact the bottom of the wells (110). In some embodiments,the cylinders have vertical freedom or movement, horizontal freedom ofmovement, or a combination of the two. The device may be configured foruse with 6, 12, 24, 36, 96, 384, or 1536 multiwell plates. It will berecognized that the device (as with the other devices and apparatusesdescribed above) may also be configured for use with multiwell plateshaving other numbers of wells (e.g., between about 6 and about 10,000wells or more).

Referring to FIG. 51, the present invention also provides a device (100)for facilitating the seeding of cells in wells of a multiwell plate. Insome embodiments, the device (100) comprises an insert (105) sized to beinserted into a well (110). The insert (105) comprises a substantiallycircular surface (115) having therein an opening (120). When the insert(105) is placed into a well (e.g., 110), the circular surface (115)extends substantially to the sidewall (125) of the well (110). Theopening (120) exposes the bottom surface (130) of the well (110) so thatwhen cells (135) are placed in the well (110) they can attach to thebottom surface (130) of the well (110). In some embodiments, the insert(105) further comprises a lift piece (140) so that the insert (105) canbe removed from the well (110). The lift piece can be configured inseveral ways, including as a tab (shown) or as an indent (not shown).When the lift piece is removed, the seeded cells (135) are confined to apredetermined area (145) of the well bottom surface (130). Thepredetermined area (145) shown is circular, however, the predeterminedarea can be any shape and located throughout the bottom surface (130) ofthe well (110). In preferred embodiments, the inserts (105) areconfigured for use with commercial 6, 12, 24, 96, 384 or 1536 wellplates. It will be recognized that the inserts can be configured for usewith custom plates and plates with non-circular wells (e.g., oval orrectangular wells).

Referring to FIGS. 55-60, the present invention further providesadditional embodiments of inserts for seeding cells in a particularpredetermined area in a well in a multiwell well plate. In someembodiments, the cell seeding insert is formed from a pliable material.In some embodiments, the cell seeding insert is formed from a polymericmaterial. In some embodiments, the cell seeding insert is formed from anelastomeric material. In particularly preferred embodiments, the cellseeding inserts are formed from silicone or PDMS. In some embodiments,the insert is formed from a rigid material. In still furtherembodiments, the insert comprises both rigid and pliable materials thatcould be formed by lamination, co-extrusion, overmolding, ormechanically affixed processes. In some embodiments, the cell seedinginserts are configured to be insertable into 6, 12, 24, 96, 384 or 1536well plates. In some embodiments, when the cell seeding insert 100 isinserted into a well in a multiwell plate (not shown), the sides of thecell seeding insert contact the sides of the well in the multiwellplate. Referring to FIGS. 55 and 59, a cell seeding insert 100 of thepresent invention is preferably cylindrical in shape, although the shapecan be varied to correspond to virtually any shape of well (square,rectangular, hexagonal, oval, etc.). In preferred embodiments, the cellseeding insert has a first end 105 and a second end 110. In someembodiments, the cell seeding insert has at least one channel therein.In some embodiments, the channel extends from an opening 120 in thefirst end of the cell seeding insert to an opening 125 in the second endof the cell seeding insert so that a fluid can be delivered from thefirst end of the cell seeding insert to the second end of the cellseeding insert when the cell seeding insert is inserted in a well of amultiwell plate (not shown). In some embodiments, the cell seedinginsert 100 further comprises a projection 130 extending from the secondend 110 of the cell seeding insert 100. In some embodiments, theprojection 130 is cylindrical in shape (i.e., as shown in FIG. 55),although in other embodiments, the projection can be any desired shapeas a square, triangle, rectangle, star, or crescent, as shown in FIG.59.

FIG. 57 provides yet another embodiment of a cell seeding insert of thepresent invention. Referring to FIG. 57, in some embodiments, the cellseeding insert 100 is cylindrical in shape and has a first end 105 andsecond end 110. In some embodiments, a channel 115 extends from thefirst end 105 of the cell seeding insert 100 to the second end 110. Thefirst and seconds ends 105 and 110 each have openings 115 thereindefining the ends of the channel 115. As above, in some embodiments, thecell seeding insert is formed from a pliable material. In particularlypreferred embodiments, the cell seeding inserts are formed from siliconeor PDMS. In some embodiments, the cell seeding inserts are configured tobe insertable into 6, 12, 24, 96, 384 or 1536 well plates. In someembodiments, when the cell seeding insert 100 is inserted into a well ina multiwell plate (not shown), the sides of the cell seeding insertcontact the sides of the well in the multiwell plate.

In some embodiments, one or more of the cell seeding inserts (e.g., thecell seeding inserts described in FIGS. 55, 57, and 59) are insertedinto one or more wells of a multiwell plate so that either theprojection on the second end of the insert (see, e.g., FIGS. 55 and 59)or the second end (see, e.g., FIG. 57) contacts the bottom of the one ormore wells of the multiwell plate. In some embodiments, cells in mediaare then seeded in the one or more wells via the channels in theinserts. In preferred embodiments, the cells seed in a predeterminedarea in the well defined as the area that is not contacted by theprojection or second end of the insert. In other words, contacts of theprojection or second end of the inserts with the bottom of the welldefine an area in which cells are excluded when cells are introducedinto the well. The cells seed in the area of the well where there is nocontact between the projection of second end of the insert.

Examples of the seeding patterns obtainable with the cell insertsdescribed in FIGS. 55, 57, and 59 are provided in FIGS. 56, 58, and 60.FIGS. 56, 58, and 60 depict the seeding pattern in the bottom of a well.Referring to FIG. 56, when the cell seeding insert of FIG. 55 isutilized, the cells are seeded in a predetermined annular area 200 andexcluded from the circular area 205 in the center of the well. Referringto FIG. 58, when the cell seeding insert of FIG. 57 is utilized, thecells are seeded in a predetermined circular area 200 in the center ofthe bottom of the well and excluded from the annular area 205 a theperiphery of the bottom of the well. Referring to FIG. 60, when the cellseeding insert of FIG. 59 is utilized, the cells are seeded in apredetermined crescent-shaped area 200 and excluded from the area 205 inthe bottom of the well.

Another cell seeding insert is depicted in FIG. 64. Referring to FIG.63, a strip of four cell seeding inserts is provided. Alternatively,strips of 6, 12, 16 or more cell seeding inserts may be provided. Thecell seeding insert preferably comprises one or more insert tips (61A),each comprising a cell exclusion tip (61B). The cell exclusion tippreferably seals with the well bottom and forms a restricted area inwhich cells are prevented from seeding. On one end, the cell exclusiontip comprises a sealing surface (61C) that contacts the well bottom. Thesealing surface preferably has therein a recessed dimple that aids insealing to the bottom of a well. As shown the cell seeding insert alsohas therein a seeding channel (61E) running the length of the insert tofacilitate adding a solution containing cells to a well in a multiwellplate. In some embodiments, where strips of inserts are provided, theinserts are separated by a hinge region 61D. The hinge region hastherein a slot on the underside (not shown) that reduces strain on theinsert backbone (61G) of the strip from one insert tip to the next. Thehinge can be severed to allow the insert tips to function as fourindividual inserts rather than as a strip. In some embodiments, theinserts further comprise a removal tool pocket (61F). The removal toolpockets are preferably angled pockets designed to interact with aremoval tool (described in more detail below). The pockets provide a gapbetween the top of the well and the bottom of the insert backbone. Theinsert backbone (61G) is a sheet of pliable material (preferablysilicone) that connects the individual inserts.

In use, the inserts are placed into the wells of a 96-well plate,oriented with the cell exclusion tips downward. Sufficient pressure isapplied to each insert to induce a seal between the sealing surface ofthe cell exclusion tips and the bottom of the well. Biological cells,suspended in media, are introduced into the wells via the seedingchannels on the side of the insert tips by using a single ormulti-channel pipette. As the cells settle to the bottom of the well,they are restricted from the center of the well by the cell exclusiontip and permitted to access to an annular region of the well. The seededplate is incubated for a period of time to allow adhesion of the cellsto the plate bottom. When the inserts are removed, the adhered cells aresituated only in an annular ring, while the center region of the wellremains void of cells. During further incubation, the biological cellsare permitted to migrate into the central, analytical zone of the well.The migration can either be monitored visually by using a microscope, orby staining the cells and then measuring absorbance of the stain byusing a plate reader. The latter method was used to seed HT1080 cellsand to observe their migration. Briefly, 50,000 cells were delivered towells of a 96-well plate that was populated with inserts. The plate wasincubated for 4 hours at 37° C. and 5% humidity to allow adherence ofcells. Following incubation, the inserts were removed and the wells werewashed with media to remove any non-adhered cells. The wells thenreceived 100 μl of media (MEM containing 10% FBS) and the plate wasincubated for an additional 21 hours to allow cell migration. The cellswere then stained with a fluorescent dye, Calcein AM, and the pattern offluorescence signal was observed by using an Axiovert microscope fittedwith a FITC filter.

In some further preferred embodiments, the present invention provides aseries of inserts in the form of a strip. In some embodiments, theindividual inserts are detachably connected to one another so thatindividual inserts can be removed from the strip. For example, in someembodiments, the inserts extends from a planar strip that hasperforations between each insert.

It will be appreciated that cell seeding inserts described above, aswell as the assay components described herein, can be provided as partof systems and kits for assaying cell migration. In preferredembodiments, these systems and kits include multiwell plates and theinserts are configured to be inserted into the multiwell plates. Infurther embodiments, the kits of the present invention includeinstructions for conducting cell migration assays. In some embodiments,the instructions further comprise the statement of intended use requiredby the U.S. Food and Drug Administration (FDA) in labeling in vitrodiagnostic products. The FDA classifies in vitro diagnostics as medicaldevices and requires that they be approved through the 510(k) procedure.Information required in an application under 510(k) includes: 1) The invitro diagnostic product name, including the trade or proprietary name,the common or usual name, and the classification name of the device; 2)The intended use of the product; 3) The establishment registrationnumber, if applicable, of the owner or operator submitting the 510(k)submission; the class in which the in vitro diagnostic product wasplaced under section 513 of the FD&C Act, if known, its appropriatepanel, or, if the owner or operator determines that the device has notbeen classified under such section, a statement of that determinationand the basis for the determination that the in vitro diagnostic productis not so classified; 4) Proposed labels, labeling and advertisementssufficient to describe the in vitro diagnostic product, its intendeduse, and directions for use. Where applicable, photographs orengineering drawings should be supplied; 5) A statement indicating thatthe device is similar to and/or different from other in vitro diagnosticproducts of comparable type in commercial distribution in the U.S.,accompanied by data to support the statement; 6) A 510(k) summary of thesafety and effectiveness data upon which the substantial equivalencedetermination is based; or a statement that the 510(k) safety andeffectiveness information supporting the FDA finding of substantialequivalence will be made available to any person within 30 days of awritten request; 7) A statement that the submitter believes, to the bestof their knowledge, that all data and information submitted in thepremarket notification are truthful and accurate and that no materialfact has been omitted; 8) Any additional information regarding the invitro diagnostic product requested that is necessary for the FDA to makea substantial equivalency determination. Additional information isavailable at the Internet web page of the U.S. FDA. It will be furtherrecognized that the cell seeding inserts can be used in methods,systems, and kits which utilize a variety of detection methods,including but not limited to colorimetric, fluorimetric, lightscattering, liquid crystal, densitometric, and microscopic assays.

In further embodiments, the present invention provides masks for usewith multiwell plates. In some embodiments, the masks are designed tomask a predetermined portion of one or more wells of a multiwell plate.In some preferred embodiments, the masks are used in conjunction withthe cell seeding inserts described above. In some embodiments, the masksare used to mask a predetermined portion of a well, wherein thepredetermined portion corresponds to an area where cells have beenseeded in a well in a multiwell plate. In such a system, the migrationof cells from the predetermined, masked portion of the well to anunmasked portion of the well can be assayed simply by determining thepresence of cells in the unmasked portion of the well. It will befurther recognized that the masks can be used in methods, systems, andkits which utilize a variety of detection methods, including but notlimited to colorimetric, fluorimetric, light scattering, liquid crystal,densitometric, and microscopic assays. The masks can also be utilized inmethods, systems, and kits that include the cell seeding insertsdescribed above.

Referring to FIG. 61, a mask (100) of the present invention is provided.In some embodiments, the mask has therein a series of openings (105)corresponding to a predetermined area within the well of a multiwellplate. In some embodiments, the mask (100) comprises a surface (110)having an adhesive so that the mask can be fixed to a multiwell plate.In some embodiments, the mask comprises a series of strips thatcorrespond to rows of wells in a multiwell plate. FIG. 61 is a depictionof one such strip. In some embodiments (not shown), the strips areattached to one another, for example, by perforations in the material ofthe mask, so that the strips may be separated and used separately tomask individual wells or rows of wells in a multiwell plate or be lefttogether and used to mask all of the wells of a multiwell plate. In someembodiments, the masks are formed from plastic. In other embodiments,the mask is made of paper or paper with a plastic coating. It will berecognized that the openings 105 in the mask 100 can be virtually anyshape, including, but not limited to circles, squares, rectangles,triangles, stars, annular rings (e.g., donut shaped with an annularopening surrounding a solid center connected to the rest of the maskedby a small extension), and so forth. In the embodiment depicted in FIG.61, the openings are preferably configured to correspond in size to thecircular area 205 in FIG. 56. In such a system, the movement of cellsseeded in the predetermined annular area 200 of FIG. 56 into thepredetermined circular area 205 can be determined.

In still further embodiments, the mask 100 has one or more aperturesassociated with and separate from the openings 105. The aperture ispreferably located so that it exposes cells initially seeded in the wellof the multiwell plate. The aperture is preferably large enough toprovide a signal that exceeds the threshold level of detection of platereader, for example, when cells are labeled with a fluorescent probe andexposed to the appropriate wavelength of excitation radiation. Thisembodiments is especially useful when plate readers are used for signaldetection and/or quantitation because by providing for a threshold levelof signal via the aperture, the migration of one or a few cells into thepredetermined, unmasked area can be detected, even if the number ofcells and signal obtained therefrom would otherwise be beneath thethreshold level of detection.

In other embodiments (not shown), the mask is sized to correspond to thesize of a multiwell plate so that the mask can be attached to theunderside (i.e., the side on which the bottom of the wells are located)of a multiwell plate. In some, the mask includes clips so that it can beattached to a multiwell plate. In other embodiments, the multiwell platecomprises clips for attachment of the mask. In still other embodiments,the multiwell plate comprises channels into which the mask can beinserted. In other embodiments the multiwell plate and the mask areattached by friction-fitting. In preferred embodiments, the maskincludes openings corresponding to a predetermined portion of thebottoms of the wells in the multiwell plate. It will be recognized thatthe openings in the mask can be virtually any shape, including, but notlimited to circles, squares, rectangles, triangles, stars, annular rings(e.g., donut shaped with an annular opening surrounding a solid centerconnected to the rest of the masked by a small extension), and so forth.In some embodiments, the openings are preferably configured tocorrespond in size to the circular area 205 in FIG. 56. In such asystem, the movement of cells seeded in the predetermined annular area200 of FIG. 56 into the predetermined circular area 205 can bedetermined. The masks can be formed from any suitable material,including, but not limited to, plastic, paper, cardboard, andplastic-coated paper or cardboard.

Another mask is the present invention is depicted in FIGS. 64 A and B.In preferred embodiments, the masks are used for cell migration assays.The mask preferably comprises of a sheet of material that fits onto thebottom of a 96-well tissue culture plate (“plate”). The mask includes 96chamfered apertures configured in an 8×12 array that correspond to thecenters of the wells in the plate. The locations of the apertures alsomatch the locations of the insert tips that populate the plate. Thechamfers function to maximize light transmission and eliminate shadowswhen the plate and mask assembly is placed on a light source. Thepurpose of the mask is two-fold. First, it blocks any signal, i.e.,emitted or transmitted light, from the biological cells that are seededin the annular region. Second, it permits the passage of signal fromcells that reside in the analytical zone. The outcome is that only cellsthat have migrated from the annular region into the analytic zone willbe detected.

Referring to FIGS. 65 and 66 A, B, C and D, the optical mask comprisesof an opaque sheet containing 96 chamfered apertures (FIG. 64A). Theapertures are configured in an 8×12 array that correspond with the wellsof the 96-well plate. The mask features five asymmetrically-placedattachment lugs (FIG. 64B) that are used to secure the mask to thebottom of the plate. The holes in the lugs fit over bosses on the bottomof the plate, establishing proper alignment. The lugs are slotted topermit them to expand slightly and engage the boss securely. The maskalso features two angled corners (64C) that mimic the profile of theplate bottom. This provides a visual cue for proper mask orientation.When the mask is fitted to the plate bottom, FIGS. 65A-D, the aperturesalign with the analytic zone in each well as established by the cellseeding inserts.

Referring to FIG. 62, in some embodiments, the present inventionprovides devices and systems for seeding cells on a slide. In someembodiments, the system 50 comprises a support 100, which in preferredembodiments is a microscope slide. The system further comprises a base105 having a series of base openings 110 therein and a channel 115extending down the long sides 120 of said base 105. Preferably, the baseopenings are circular, although the openings may be any other desiredshape. In some embodiments, a gasket 125, preferably formed from apliable material, is disposed between the support 100 and the base 105.The gasket 125 preferably has a series of gasket openings (not shown)therein, alignable with the base openings 110 in the base 105. The base,gasket, and support may be aligned and held together by any means knownin the art, including spring clips, polymeric clips, and through the useof adhesive strips. In some preferred embodiments, the base, gasket andsupport are held together by two clips 130. The clips preferablycomprise channel extensions 135 that engage the channels 115 in the base105. The clips further comprise a support extension 140 that engages thesupport 100.

In further preferred embodiments, the present invention provides aninsert removal tool to be used in conjunction with the well inserts ofthe present invention, referring to FIG. 63, the insert removal tool 100comprises an insert end 105 comprising a plurality of extensions 110.The extensions 110 are separated by openings 115. The extensions 110 aresized so that the extension may be inserted between inserts located in amultiwell plate so that the extensions extend past the sides of theinsert and the inserts are thus located in the openings 115. The insertremoval tool 100 further comprises a handle 120 extending from theinsert end 105. In use, the insert end is applied to multiwell platecontaining inserts so that the extensions extend on either side ofinserts located in wells in of the multiwell plate. Pressure can then beexerted on the handle to extract inserts from the plate. In preferredembodiments, the handle 120 extends away from the extension end 105 atapproximately a 90 degree angle so that the handle can be pulledvertically away from the plate to exert pressure on the inserts, thusfreeing the inserts from the plate.

In preferred embodiments, the present insert removal tool is laser cutand subsequently formed stainless steel implement. The insert removaltool preferably comprises two elements, the handle and the head. Thehead is formed by bending the distal end of the cut metal at threeplaces to establish a ‘goose neck’ configuration. The distal end of thehead is positioned 90° to the handle and terminates in five individualtines. The recesses between the tines form pockets which engage andsupport the insert tips during extraction. The gooseneck feature on thehead permits the handle to align with the center line of the insertbackbone when the tool is engaged with the insert. The tool is used bysliding the tines beneath the insert backbone until the tool and insertare fully engaged. The removal tool is then lifted vertically,extracting the cell seeding insert without disturbing the neighboringinserts.

EXPERIMENTAL Example 1 Fabrication of Nanostructured Substrates for Usein Cell Assays

Nanostructured substrates that align liquid crystal (LC) for use in LCcell assays have been fabricated using: 1) nanoscale molding, 2)nanoabrasives, 3) obliquely deposited gold films.

Nanomolded substrates were fabricated by the molding of polyurethane andpolystyrene from hard masters prepared in silicon. The silicon masterswere prepared by electron beam lithography and the topography present inthe masters had a width of 200 nm and a height of 50 nm. Studiesindicated that immersion of these substrates into aqueous media(phosphate buffered saline) for at least 24 hours does not lead to lossof topography. Furthermore, because of the relatively large scalefeatures, these substrates did not respond (in terms of LC alignment) tonon-specific protein adsorption. Polyurethane, micromolded,nanostructured substrates with 50 nm deep tracks were incubated for 4hrs with cell culture medium (MEM) containing 10% fetal bovine serum.The ability to align LC's was not affected. When viewed with apolarizing microscope, the area within the tracks appeared dark anduniform, while the areas outside of the tracks appeared bright. Sharplateral resolution of the LC alignment between nanostructured regionsand non-structured region of the surface was observed microscopically.

Abraded surfaces were created on glass slides by hand rubbing the slideswith a fine commercial emery cloth. Even pressure was applied throughoutthe rubbing. A liquid crystal film was applied on the surface of theabraded slide and viewed through polarizing filters. The liquid crystalfilm appeared dark when viewed through crossed polars indicating thatthe liquid crystals were aligned on the nano-abraded surface. Cellculture medium (MEM) containing 10% fetal calf serum was incubated onthe surfaces. A film of LC's was added to the surface. When viewed withpolarizing lenses, the LC's were aligned on the surface, indicating thatthe alignment of LC's is not affected by protein adsorption to thenano-abraded surface.

Obliquely deposited gold films were prepared on glass slides. The goldwas deposited from a vapor that impacts the glass slide at an obliqueangle of incidence (12.5 degrees). This method leads to a surface withnano-meter scale statistical topography. The slide was incubated withcell culture medium (MEM) containing 10% fetal calf serum. Following theremoval of the cell culture medium, a layer of LC was applied and theslide was visualized through polarizing filters. The liquid crystalswere aligned on the slide. This demonstrates that such surfaces will beuseful in liquid crystal based cellular assays.

Example 2 Liquid Crystals Reliably Report Cell Number

CHO K1 cells were seeded in DMSO onto anisotropically orderednanostructured substrates of obliquely deposited gold that align liquidcrystals. The cells were allowed to attach for 4 hours. The media wasremoved and the cells were fixed in methanol, gluteraldehyde or theywere left unfixed. The slides were either dried in a stream of Nitrogenor left wet and then covered with a film of a nematic LC. A variety ofLC films, including 5CB, E7 and TL205 were able to accurately report thepresence of cells regardless of their state of hydration or method offixation. Substrates with 100, 300, 600 and 1200 cells per 3.14 mm2(equivalent to approx 5,600-67,000 cells seeded into a single 15 mmdiameter of a 24 well plate) were imaged through polarizing filters andthe images were analyzed for gray scale intensity using NIH Imagesoftware. The presence of cells locally prevents the LC film fromgaining access to the ordering influence of the underlyingnanostructured substrate. Regions of the LC film directly overlyingcells are therefore disordered and do not preserve the plane ofpolarization of light. When viewed in a cross polar configuration theamount of light transmitted is therefore proportional to cell number.These data are presented in FIG. 47.

Example 3 Stability of LC Response

It was noted in the experiments conducted with cells, the LC assaysprovide an unambiguous signal that persists for weeks withoutappreciable change. This was true of cells that were fixed prior to theaddition of LC and to live cells coated with the LC.

Example 4 LC's do not Inhibit Cell Mobility

Matrigel (diluted 1:1 with MEM) and LC TL 205 were mixed in equalvolumes and the emulsion was applied to a glass slide which wasincubated at 37 C for 30 minutes. Hepa-1c1c7 (hepatic rodent cell line)was plated onto the matrix and incubated for 2 hrs. The cells werevisualized with a microscope. In the presence of LC TL 205, the cellsformed tracks on the Matrigel. This experiment shows that LC 205 is notinhibitory to cellular movement and that imaging of cellular movement onMatrigel by LC's is feasible.

Example 5 Quantification of an Analyte Using Microfluidic Channels andLiquid Crystals

This example describes the results of an experiment in which disruptionof a liquid crystal along a microfluidic channel was used quantify theamount of an analyte in a sample. Five microchannels were (1 mm wide by25 μm deep) were formed on a block of PDMS that was supported on a glassslide. Samples containing PBS (control) or biotin-bovine serum albumin(BSA) in varying concentrations: 12.5, 25, 50 or 100 μg/ml, were placedinto a reservoir and moved into the microchannel by capillary force. Thesubstrate was incubated at 37° C. for 60-90 minutes. After incubation, avacuum was applied to remove the liquids from the microchannels andreservoirs. The PDMS was then peeled off from the glass slide, rinsedwith water, dried in a stream of nitrogen gas and placed onto an OTStreated glass slide. Liquid crystals (E7) were introduced into eachmicrochannel through its reservoir by capillary force. Images of thealignment of liquid crystals inside the microchannels were taken with apolarized microscope.

Liquid crystals assume a homeotropic alignment on the PDMS in theabsence of bound protein. This region appears dark and homogenous whenviewed through polarizing lenses. When biotin-BSA is present on thesurface of the microchannel, the liquid crystal loses its homeotropicalignment. This disruption is evidenced by the transmission of light inthe regions where protein is bound. The higher the concentration ofprotein in the sample, the longer the region of disruption along themicrochannel (FIG. 48). The length of the disruption of liquid crystalsalignment in the microchannel was measured from the images by using thewidth of the microchannel (1 mm) as a standard. Additional experimentswith higher concentrations of biotin-BSA (6.25-400 μg/ml) showed similarresults.

Example 6 Detection of Zymographic Activity by Liquid Crystals

The enzymatic digestion of a protein substrate can be detected by theorientation of a layer of liquid crystals placed upon the substratefollowing exposure to an enzyme. To demonstrate this, we prepared thinfilms of obliquely deposited gold (45°) on glass slides. The gold slidewas functionalized with a self assembling monolayer of an alkanethiol (2mM, C16SH). A solution of collagen IV (BD Biosciences, 50 μg/mL) wasincubated on the slide for 2 hrs at 37° C. A test sample containing 1 pgtotal enzyme of activated MMP-9 (Chemicon) in 5 mM Tris buffer with0.005 IGEPAL, pH7.5 was added to the surface and incubated for 30minutes at 37° C. The surfaces were washed with water, dried with astream of nitrogen and a drop of liquid crystal was placed on thesurface. The alignment of the liquid crystal was observedmicroscopically and the degree of disruption of the liquid crystalalignment was measured using Scion software.

Liquid crystals align in a regular fashion on the surfaces prepared withSAM. On surfaces presenting collagen IV, the liquid crystals appeardisordered when viewed through a polarizing filter. If MMP-9 digests thecollagen IV, the underlying SAM is exposed and liquid crystals align onthe SAM. FIG. 49 demonstrates that a surface presenting collagen IVcauses disruption in the alignment of the liquid crystal layer (0 min),however, after 30 minutes incubation with 1 pg MMP-9, the collagen IVhas been digested and the liquid crystals align on the exposed SAM (30min).

Example 7 Homeotropic Orientation by Cells

Tables 2 and 3 present the results of experiments in which differentliquid crystals were surveyed for their ability to be homeotropicallyoriented by cultured cells. Many liquid crystals align homeotropicallyin response to phospholipids and cholesterol. Phospholipids (2 ul; 0.01M in chloroform) were applied to discrete marked areas on glass slides.

TABLE 2 Survey of liquid crystals for alignment by cells and by slideexposed to medium. Liquid Crystal FBS/DMEM 3T3 cells 4OCB DisruptedHomeotropic 5CB Disrupted Homeotropic 6CHBT Planar, with defectsHomeotropic E7 Disrupted Homeotropic ZLI-1221 Planar, streaky DisruptedZLI-1557 Planar with streaky defects Homeotropic ZLI-2222 Planar, minordefects Homeotropic ZLI-3225 Planar with streaky defects Homeotropic(tilt) ZLI-3497 Planar with streaky defects Homeotropic (tilt) ZLI-4431Planar with streaky defects Homeotropic (tilt) ZLI-4446 Planar, withdefects Homeotropic ZLI-5070 Planar with streaky defects Homeotropic(tilt) MLC-6080 Planar with squiggly defects Homeotropic MLC-6466 Planarwith streaky defects Homeotropic MLC-6710-080 Planar with streakydefects Homeotropic MLC-15700-000 Planar, streaky Homeotropic TL205Somewhat planar HomeotropicThe phospholipids had dioleoyl alkyl chains and the followingheadgroups: phosphatidylserine (DOPS), phosphatidylglycerol (DOPG),phosphatidylethanolamine (DOPE), phosphatidylserine (DOPS), phosphatidicacid (DOPA), and lysophosphatidylcholine (DOLPC). After the solventdried, optical cells were assembled with liquid crystals appliednematically and heated to isotropy. Homeotropic alignment was confirmedby conoscopic analysis. Chol=cholesterol; C=cholesteric alignment;Bkg=background alignment; U=unaligned; H=homeotropically aligned; NDindicates not done due to background. 40CB,4′-octyl-4-biphenyl-carbonitrile (Aldrich); 6CHBT,1-(trans-4-hexylcyclohexyl)-4-isothiocyanato-benzene. All other liquidcrystals are from EM Industries/Merck.

TABLE 3 Investigation of phospholipid influence on liquid crystalalignment. Liquid Crystal Bkg DOPS DOPG DOPC DOPE DOPA DOLPC Chol 4OCB HND ND ND ND ND ND ND 5CB U H H H Planar H U 6CHBT U H H H H H U E7Twisted H H H Twisted H H planar planar ZLI-1221 H H H H H H H ZLI-1557H H H H H H H ZLI-2222 H H H H H H H ZLI-3225 U H H H H H H ZLI-3497 HND ND ND ND ND ND ND ZLI-4431 Chol U U U U U U U ZLI-4446 H ND ND ND NDND ND ND ZLI-5070 Twisted H H H H H H planar MLC-6080 U H H H H H HMLC-6466 U H H H H H H MLC-6710-080 U H H H H H H MLC-15700-000 H H H UH H U TL205 U H H H H H H

Example 8 Demonstration of Mask Functionality

A 100 ul portion of 3T3 fibroblasts (at 25,000 cells per well andtreated with mitomycin C to inhibit proliferation) were seeded intowells of a Greiner 96-well flat bottom plate that contained cell seedinginserts. The fibroblasts were allowed to adhere for four hours at 37°C., 5% CO₂. The inserts were then removed from the test wells and thewells were washed with PBS to remove non-adhered cells. A 100 ul volumeof cell culture media (MEM containing 10% FBS) was then introduced intoeach well to promote growth. In negative control wells, the seedinginserts remained in place for the duration of the incubations. Theseeded plate was incubated overnight (˜21 hours) to permit migration ofthe cells in the test wells. Following incubation, the inserts wereremoved from the control wells. All wells were washed with PBS and thecells were stained with a fluorescent Calcein AM dye using standardmethods per manufacturer instructions. The well contents were observedby using a Zeiss Axiovert microscope (2.5× objective, FITC filter) anddigital images were captured both in the absence and presence of themask.

The amount of fluorescent signal was quantified by use of a platereader. Briefly, the plate was inserted into the Bio-Tek Synergy platereader and fluorescence signal was measured by using parameters thatincluded 528/533 nm wavelength, a gain sensitivity of 55, and a bottomprobe read. The relative fluorescence units (RFUs) were captured withthe mask in place for both the control and test wells (N=8 replicatesper condition). The RFU data was subjected to a 5PLE calculation thatconverts signal into numbers of cells detected. The results of thisstudy indicated that the fluorescence signal in the central zone of thetest wells represented 240+/−37 cells while that signal in the controlwells represented 29+/−8 cells (data not shown).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in chemicalengineering, cell biology, or molecular biology or related fields areintended to be within the scope of the following claims.

1. A device for seeding cells in a well in a multiwell plate comprising:an insert sized to be inserted into a well of a multiwell plate, saidinsert having a first end and a second end and having at least onechannel therein extending from said first end to said second end, saidsecond end having an opening in fluid communication with said channeland comprising a solid projection extending from said second end, saidsolid projection having a perimeter smaller than the perimeter of saidinsert so that said channel opening is outside of said solid projection;wherein when said device is inserted into a well of a multiwell plate,said solid projection extending from said second end contacts the bottomof said well to seal off a portion of the bottom of said well and saidopening provides fluid access to the bottom of said well wherein cellscan be delivered to the bottom of said well via said at least onechannel.
 2. The device of claim 1, wherein said insert is cylindrical.3. The device of claim 1, wherein said projection is circular in shapeso that when cells are delivered to said well, the cells seed in anannular pattern in which cells are absent from the center of said well.4. The device of claim 1, wherein said projection is shaped to provide acrescent-shaped opening.
 5. The device of claim 1, comprising a channelthrough the interior of said insert, wherein said channel provides saidopening in said second end.
 6. The device of claim 5, wherein saidopening in said second end is circular.
 7. The device of claim 1,wherein said projection is formed from a material selected from thegroup consisting of PDMS and silicone.
 8. The device of claim 1, whereinsaid device is sized to be inserted into a well of a multiwell plateselected from the group consisting of 6, 12, 24, 96, 394, and 1536 wellplates.
 9. The device of claim 1, wherein said device has two channelstherein that extend from said first end to said second end.
 10. Asystem, comprising a multiwell plate; at least one insert sized to beinserted into a well of a multiwell plate, said insert having a firstend and a second end and having at least one channel therein extendingfrom said first end to said second end, said second end having anopening in fluid communication with said channel and comprising a solidprojection extending from said second end, said solid projection havinga perimeter smaller than the perimeter of said insert so that saidchannel opening is outside of said projection; wherein when said insertis inserted into a well of a multiwell plate, said solid projectionextending from said second end contacts the bottom of said well to sealoff a portion of the bottom of said well and said opening provides fluidaccess to the bottom of said well wherein cells can be delivered to thebottom of said well via said at least one channel.
 11. The system ofclaim 10, wherein said insert is cylindrical.
 12. The system of claim10, wherein said projection is circular in shape so that when cells aredelivered to said well, the cells seed in an annular pattern in whichcells are absent from the center of said well.
 13. The system of claim10, wherein said projection is shaped to provide a crescent-shapedopening.
 14. The system of claim 10, wherein said insert comprises achannel through the interior of said insert, wherein said channelprovides said opening in said second end.
 15. The system of claim 14,wherein said opening in said second end is circular.
 16. The system ofclaim 10, wherein said projection is formed from a pliable material. 17.The system of claim 10, wherein said device is sized to be inserted intoa well of a multiwell plate selected from the group consisting of 6, 12,24, 96, 394, and 1536 well plates.
 18. The system of claim 10, whereincomprising a plurality of said inserts, wherein said inserts areprovided in a strip and wherein said individual inserts are detachablyconnected.